Microbial compositions for fowl health and performance

ABSTRACT

The disclosure relates to isolated microorganisms—including novel strains of the microorganisms—microbial ensembles, and compositions comprising the same. Furthermore, the disclosure teaches methods of utilizing the described microorganisms, microbial ensembles, and compositions comprising the same, in methods for modulating the production of poultry, disease resistance, and egg yield. In particular aspects, the disclosure provides methods of increasing feed efficiency, and methods of preventing colonization of pathogenic microbes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application under 35 U.S.C. §371 of International PCT Application No. PCT/US2018/056563, filed onOct. 18, 2018, which claims priority to and benefit of U.S. ProvisionalApplication No. 62/574,031, filed on Oct. 18, 2017; each of which areincorporated herein by reference in their entirety.

FIELD

The present disclosure relates to isolated and biologically puremicroorganisms that have applications, inter alia, in the farming offowl or poultry. The disclosed microorganisms can be utilized in theirisolated and biologically pure states, as well as being formulated intocompositions. Furthermore, the disclosure provides bioensembles,containing at least two members of the disclosed microorganisms, as wellas methods of utilizing said bioensembles. Furthermore, the disclosureprovides for methods of modulating the fowl or poultry microbiome tomodulate the immune system.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is ASBI_012_01WO_ST25.txt. The text file is 165 kb,was created on Oct. 17, 2018, and is being submitted electronically viaEFS-Web.

BACKGROUND

The global population is predicted to increase to over 9 billion peopleby the year 2050 with a concurrent reduction in the quantity of land,water, and other natural resources available per capita. Projectionsindicate that the average domestic income will also increase, with theprojected rise in the GDP of China and India. The desire for a dietricher in animal-source proteins rises in tandem with increasing income,thus the global livestock sector will be charged with the challenge ofproducing more animal products using fewer resources. The Food andAgriculture Organization of the United Nations predict that 70% morefood will have to be produced, yet the area of arable land availablewill decrease. It is clear that the food output per unit of resourceinput will have to increase considerably in order to support the rise inpopulation.

Over recent decades the farm industry has seen fast growth in the meatsector, which has been underpinned by rising demand for poultry meat,which has consistently increased at about three times the rate ofpopulation growth over each of the past five decades.

Poultry meat, eggs, and components thereof are predominantly utilized inthe preparation of foodstuffs in many different forms. There have beenmany strategies to improve poultry and egg production throughnutritional modulations, hormone treatments, changes in animalmanagement, and selective breeding; however, the need for more efficientproduction of edible poultry foodstuffs per animal is required.

Identifying compositions and methods for sustainably increasing poultryand egg production while balancing animal health and wellbeing havebecome imperative to satisfy the needs of everyday humans in anexpanding population. Increasing the worldwide production of poultry byscaling up the total number of poultry on farms would not only beeconomically infeasible for many parts of the world, but would furtherresult in negative environmental consequences as the poultry sector'sgrowth and trends towards intensification and concentration have alreadygiven rise to a number of environmental concerns, led predominantly bythe production of far more waste than can be managed by land disposal.

Population densities of poultry in large farms are often accompanied byan increased incidence of microbial pathogens that place the poultryyield at risk, and further place the ultimate consumer of the poultry atrisk in instances of zoonotic pathogens such as those of Clostridium andSalmonella. Considering the widespread occurrence of many zoonoticpathogens, it is unlikely that poultry can be completely protected fromexposure. Research has focused on investigative means of increasingresistance to colonization in poultry exposed to these pathogens.Furthermore, large farm poultry operations turn out large flocks thatexhibit a high degree of variability in size, health, meat content dueat least in part to the great degree of variability exhibited in thediversity of the reproductive and gut microbiomes.

Thus, meeting global poultry yield expectations, by simply scaling upcurrent high-input agricultural systems-utilized in most of thedeveloped world—is simply not feasible.

There is therefore an urgent need in the art for improved methods ofincreasing poultry and egg production, while also mitigating thecolonization and spread of microbial pathogens.

SUMMARY OF THE DISCLOSURE

In some embodiments, the disclosure is generally drawn to a method forimproving one or more desirable traits in fowl, the method comprising:(a) administering to a fowl an effective amount of a microbialcomposition comprising: (i) a purified microbial population thatcomprises one or more bacteria with a 16S nucleic acid sequence thatshares at least 97% sequence identity with a nucleic acid sequenceselected from SEQ ID NOs: 1-50 and 59-387 and/or one or more fungi withan ITS nucleic acid sequence that shares at least 97% sequence identitywith a nucleic acid sequence selected from SEQ ID NOs: 51-58, and (ii) acarrier suitable for fowl administration; wherein the purified microbialpopulation is present in the microbial composition in an amounteffective to improve the one or more desirable traits as compared to afowl not having been administered the microbial composition; and whereinthe improvement of the one or more desirable traits is an improvement inthe innate immune response, an improvement in incidence of normalgastrointestinal morphology, an improvement in growth rate, animprovement in total body mass, an improvement in feed conversion ratio,an improvement in pathogen exclusion, an improvement in competitiveexclusion against pathogens, a reduction in mortality, a reduction inflock variability, an improvement in antimicrobial production, animprovement in stimulating the production of B cells, an improvement instimulating the production of lymphocytes, an improvement in length ofvilli, and an improvement in expression of inflammatory cytokines.

In some embodiments, the fowl is a broiler. In some embodiments, the oneor more bacteria and/or the one or more fungi have a MIC score of atleast about 0.1.

In some embodiments, the purified microbial population comprises abacterium with a 16S nucleic acid sequence sharing at least 97% identitywith SEQ ID NO:386. In some embodiments, the purified microbialpopulation comprises a bacterium with a 16S nucleic acid sequencesharing at least 97% identity with SEQ ID NO:387. In some embodiments,the purified microbial population further comprises one or more bacteriawith a 16S nucleic acid sequence sharing at least 97% sequence identitywith nucleic acid sequences selected from the group consisting of SEQ IDNO:13, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:369, SEQ IDNO:374, and SEQ ID NO:348. In some aspects, the purified microbialpopulation comprises one or more bacteria with a 16S nucleic acidsequence sharing at least 97% sequence identity with SEQ ID NO:386 andSEQ ID NO:387. In some aspects, the purified microbial populationcomprises one or more bacteria with a 16S nucleic acid sequencecomprising SEQ ID NO:386 and SEQ ID NO:387. In some aspects, thepurified microbial population comprises one or more bacteria with a 16Snucleic acid sequence selected from the group consisting of SEQ IDNO:13, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:369, SEQ IDNO:374, and SEQ ID NO:348. In some embodiments the purified microbialpopulation further comprises one or more bacteria with a 16S nucleicacid sequence sharing at least 97% sequence identity with nucleic acidsequences selected from the group consisting of SEQ ID NO:1 and SEQ IDNO:3. In some embodiments, the purified microbial population comprisesone or more bacteria with a 16S nucleic acid sequence sharing at least97% sequence identity with SEQ ID NO:386 and SEQ ID NO:387. In someembodiments, the purified microbial population comprises one or morebacteria with a 16S nucleic acid sequence comprising SEQ ID NO:386 andSEQ ID NO:387. In some embodiments, the purified microbial populationcomprises one or more bacteria with a 16S nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:1 and SEQ ID NO:3.

In some embodiments, the microbial composition is formulated as anencapsulation, tablet, capsule, pill, feed additive, food ingredient,food preparation, food supplement, water additive, water-mixed additive,heat-stabilized additive, moisture-stabilized additive, pre-pelletedfeed additive, pelleted feed additive, post-pelleting-applied feedadditive, consumable solution, consumable spray additive, consumablesolid, consumable gel, injection, suppository, drench, bolus, orcombination thereof.

In some embodiments, the improvement of the one or more desirable traitsis an improvement in antimicrobial production. In some embodiments, theimprovement of antimicrobial production is an improvement ofantimicrobial production in the gastrointestinal tract. In someembodiments, the administering of a purified microbial populationcomprises administering a first microbial composition and a secondmicrobial compositions.

In some embodiments, the microbial composition elicits a decrease in thevariability of the number of unique species in the gastrointestinaltract. In some embodiments, the decrease in the variability of thenumber of unique species is a reduction of the total number of uniquespecies of microbes to between about 50 and 400 species.

In some embodiments, the disclosure is generally drawn to a microbialcomposition comprising (i) a purified microbial population thatcomprises one or more bacteria with a 16S nucleic acid sequence thatshares at least 97% sequence identity with a nucleic acid sequenceselected from SEQ ID NOs: 1-50 and 59-387 and/or one or more fungi withan ITS nucleic acid sequence that shares at least 97% sequence identitywith a nucleic acid sequence selected from SEQ ID NOs: 51-58, and (ii) acarrier suitable for fowl administration; wherein the purified microbialpopulation is present in the microbial composition in an amounteffective to improve the one or more desirable traits as compared to afowl not having been administered the microbial composition; and whereinthe improvement of the one or more desirable traits is an improvement inthe innate immune response, an improvement in incidence of normalgastrointestinal morphology, an improvement in growth rate, animprovement in total body mass, an improvement in feed conversion ratio,an improvement in pathogen exclusion, an improvement in competitiveexclusion against pathogens, a reduction in mortality, a reduction inflock variability, an improvement in antimicrobial production, animprovement in stimulating the production of B cells, an improvement instimulating the production of lymphocytes, an improvement in length ofvilli, and an improvement in expression of inflammatory cytokines; andwherein at least one of the one or more bacteria and/or at least one ofthe one or more fungi is desiccated.

In some aspects, the fowl is a broiler. In some aspects the one or morebacteria and/or the one or more fungi have a MIC score of at least about0.1.

In some embodiments, the purified microbial population comprises abacterium with a 16S nucleic acid sequence sharing at least 97% identitywith SEQ ID NO:386. In some embodiments, the purified microbialpopulation comprises a bacterium with a 16S nucleic acid sequencesharing at least 97% identity with SEQ ID NO:387. In some embodiments,the purified microbial population further comprises one or more bacteriawith a 16S nucleic acid sequence sharing at least 97% sequence identitywith nucleic acid sequences selected from the group consisting of SEQ IDNO:13, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:369, SEQ IDNO:374, and SEQ ID NO:348. In some aspects, the purified microbialpopulation comprises one or more bacteria with a 16S nucleic acidsequence sharing at least 97% sequence identity with SEQ ID NO:386 andSEQ ID NO:387. In some aspects, the purified microbial populationcomprises one or more bacteria with a 16S nucleic acid sequencecomprising SEQ ID NO:386 and SEQ ID NO:387. In some aspects, thepurified microbial population comprises one or more bacteria with a 16Snucleic acid sequence selected from the group consisting of SEQ IDNO:13, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:369, SEQ IDNO:374, and SEQ ID NO:348. In some embodiments the purified microbialpopulation further comprises one or more bacteria with a 16S nucleicacid sequence sharing at least 97% sequence identity with nucleic acidsequences selected from the group consisting of SEQ ID NO:1 and SEQ IDNO:3. In some embodiments, the purified microbial population comprisesone or more bacteria with a 16S nucleic acid sequence sharing at least97% sequence identity with SEQ ID NO:386 and SEQ ID NO:387. In someembodiments, the purified microbial population comprises one or morebacteria with a 16S nucleic acid sequence comprising SEQ ID NO:386 andSEQ ID NO:387. In some embodiments, the purified microbial populationcomprises one or more bacteria with a 16S nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:1 and SEQ ID NO:3.

In some embodiments, the microbial composition is formulated as anencapsulation, tablet, capsule, pill, feed additive, food ingredient,food preparation, food supplement, water additive, water-mixed additive,heat-stabilized additive, moisture-stabilized additive, pre-pelletedfeed additive, pelleted feed additive, post-pelleting-applied feedadditive, consumable solution, consumable spray additive, consumablesolid, consumable gel, injection, suppository, drench, bolus, orcombination thereof.

In some embodiments, the improvement of the one or more desirable traitsis an improvement in antimicrobial production. In some embodiments, theimprovement of antimicrobial production is an improvement ofantimicrobial production in the gastrointestinal tract.

In some embodiments, the microbial composition elicits a decrease in thevariability of the number of unique species in the gastrointestinaltract. In some embodiments, the decrease in the variability of thenumber of unique species is a reduction of the total number of uniquespecies of microbes to between about 50 and 400 species.

Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedures

Some microorganisms described in this application were deposited withthe United States Department of Agriculture (USDA) Agricultural ResearchService (ARS) Culture Collection (NRRL®), located at 1815 N. UniversitySt., Peoria, IL 61604, USA. Some microorganisms described in thisapplication were deposited with the Bigelow National Center for MarineAlgae and Microbiota, located at 60 Bigelow Drive, East Boothbay, Maine04544, USA. Some microorganisms described in this application weredeposited with the American Type Culture Collection (ATCC), located at10801 University Boulevard, Manassas, Virginia 20108, USA.

The deposits were made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. The NRRL®, ATCC, and Bigelow NationalCenter for Marine Algae and Microbiota accession numbers for theaforementioned Budapest Treaty deposits are provided in Table 2. Theaccession numbers and corresponding dates of deposit for themicroorganisms described in this application are separately provided inTable 15.

The strains designated in the below table have been deposited in thelabs of Ascus Biosciences, Inc. since at least Mar. 1, 2016. Strainscomprising SEQ ID NOs:386 and 387 were isolated prior to, and storedsince October 2017.

In Table 1, the closest predicted hits for taxonomy of the microbes arelisted in columns 2 and 5. Column 2 is the top taxonomic hit predicatedby BLAST, and column 5 is the top taxonomic hit for genus+speciespredicted by BLAST. The strains designated in the below table have beendeposited in the labs of Ascus Biosciences, Inc. since at least Mar. 1,2016.

Table 1 lists strain designations of the bacteria and fungi of thepresent disclosure. If a letter in parentheses follows any of the straindesignations, then that indicates that each of those strains havevariants that share at least 97% sequence identity with the referencestrain with the (A) parenthetical. Ascusbbr_5796(A) has two variants,Ascusbbr_5796(B) and Ascusbbr_5796(C) that share 97.8% and 98.2%sequence identity, respectively, with Ascusbbr_5796(A).Ascusbbr_14690(A) has two variants, Ascusbbr_14690(B) andAscusbbr_14690(C) that share 97.8% and 98.2% sequence identity,respectively, with Ascusbbr_14690(A). Ascusbbr_38717(A) shares 98.6%sequence identity with Ascusbbr_38717(B). Ascusbbr_33(A) shares 98.2%sequence identity with Ascusbbr_33(B). Ascusbbr_409(B), Ascusbbr_409(C),Ascusbbr_409(D), share 98.2%, 97.3%, and 97.8% sequence identity,respectively, with Ascusbbr_409(B). Ascus_331885(B) and Ascus_331885(C)share 97.8% and 97.3% sequence identity, respectively, withAscus_331885(A). Ascusbbr_247(A) shares 97.8% sequence identity withAscusbbr_247(B). Ascusbbr_10593(A) shares 99.6% sequence identity withAscusbbr_10593(B). Ascusbbr_32731(A) shares 97.3% sequence identity withAscusbbr_32731(B). Ascusbbr_1436(A) shares 97.8% sequence identity withAscusbbr_1436(B). Ascusbbr_265(A) shares 99.6% sequence identity withAscusbbr_265(B).

TABLE 1 Microbes of the present disclosure, including bacteria (1-99)and fungi (100-107). Se- quence BLAST Identi- Taxonomic fier forPredicted Closest BLAST BLAST Top Hit BLAST Asso- Taxa of IsolatedTaxonomic % Query w/Genus + % Query Strain ciated MIC Microbes Top HitIdent. Cover Species Ident. Cover Designation Marker Score 1.Lactobacillus Lactobacillus 98% 100% Lactobacillus 98% 100%Ascusbbr_4729 SEQ ID 0.76676 (Genus) crispatus crispatus NO: 1 2.Lachnospiraceae Bacterium ic1296 98%  91% Ruminococcus 95%  98%Ascusbbr_339 SEQ ID 0.62924 Clostridium gnavus NO: 2 Cluster XIVa(Family + Cluster) 3. Lactobacillus Lactobacillus 100%  100%Lactobacillus 100%  100% Ascusbbr_5796(A) SEQ ID 0.61325 (Genus)crispatus crispatus NO: 3 4. Lactobacillus Lactobacillus 100%  100%Lactobacillus 100%  100% Ascusbbr_5796(B) SEQ ID 0.61325 (Genus)crispatus crispatus NO: 369 5. Lactobacillus Lactobacillus 100%  100%Lactobacillus 100%  100% Ascusbbr_5796(C) SEQ ID 0.61325 (Genus)crispatus crispatus NO: 370 6. Lactobacillus Lactobacillus 99% 100%Lactobacillus 99% 100% Ascusbbr_38717(A) SEQ ID 0.59229 (Genus)vaginalis vaginalis NO: 4 7. Lactobacillus Lactobacillus 99% 100%Lactobacillus 99% 100% Ascusbbr_38717(B) SEQ ID 0.59229 (Genus)vaginalis vaginalis NO: 373 8. Lactobacillus Lactobacillus 99%  98%Lactobacillus 99%  98% Ascusbbr_170211 SEQ ID 0.58403 (Genus) vaginalisvaginalis NO: 5 9. Lactobacillus Lactobacillus 99% 100% Lactobacillus99% 100% Ascusbbr_1686 SEQ ID 0.57845 (Genus) johnsonii johnsonii NO: 610. Faecalibacterium Faecalibacterium 90%  98% Faecalibacterium 89%  97%Ascusbbr_1789 SEQ ID 0.56099 (Genus) sp. prausnitzii NO: 7 11.Lactobacillus Lactobacillus 99% 100% Lactobacillus 99% 100%Ascusbbr_3820 SEQ ID 0.55862 (Genus) johnsonii johnsonii NO: 8 12.Hydrogenoanaero Clostridium sp. 91%  98% Butyrivibrio 86%  82%Ascusbbr_173 SEQ ID 0.55675 bacterium hungatei NO: 9 (Genus) 13.Peptostrepto- Clostridium sp. 92% 100% [Eubacterium] 91%  98%Ascusbbr_3089 SEQ ID 0.55548 coccaceae tenue NO: 10 Clostridium ClusterXI (Family + Cluster) 14. Acrocarpospora Nonomuraea sp. 99%  88%Microbispora 95% 100% Ascusbbr_167 SEQ ID 0.5442 (Genus) rosea NO: 1115. Lactobacillus Lactobacillus 99%  98% Lactobacillus 99%  98%Ascusbbr_301568 SEQ ID 0.53873 (Genus) helveticus helveticus NO: 12 16.Bacillus (Genus) Bacillus 99% 100% Bacillus 99% 100% Ascusbbr_33(A) SEQID 0.53686 subtilis subtilis NO: 13 17. Bacillus (Genus) Bacillus 99%100% Bacillus 99% 100% Ascusbbr_33(B) SEQ ID 0.53686 subtilis subtilisNO: 374 18. Lactobacillus Lactobacillus 99%  95% Lactobacillus 99%  95%Ascusbbr_25200 SEQ ID 0.52435 (Genus) coleohominis coleohominis NO: 1419. Subdoligranulum Bacterium ic1340 90%  98% Anaerofilum 88%  91%Ascusbbr_84 SEQ ID 0.52174 (Genus) pentosovorans NO: 15 20.Subdoligranulum Firmicutes 99% 100% Faecalibacterium 96% 100% Ascusbbr136 SEQ ID 0.51373 (Genus) bacterium prausnitzii NO: 16 21.Lachnospiraceae Clostridium sp. 95% 100% Eubacterium 94%  98%Ascusbbr_128 SEQ ID 0.51348 Clostridium fissicatena NO: 17 Cluster XIVa(Family + Cluster) 22. Lactobacillus Lactobacillus 99%  95%Lactobacillus 99%  95% Ascusbbr_322104 SEQ ID 0.50724 (Genus)coleohominis coleohominis NO: 18 23. Lactobacillus Lactobacillus 100% 100% Lactobacillus 100%  100% Ascusbbr_409(A) SEQ ID 0.50572 (Genus)reuteri reuteri NO: 19 24. Lactobacillus Lactobacillus 100%  100%Lactobacillus 100%  100% Ascusbbr_409(B) SEQ ID 0.50572 (Genus) reuterireuteri NO: 375 25. Lactobacillus Lactobacillus 100%  100% Lactobacillus100%  100% Ascusbbr_409(C) SEQ ID 0.50572 (Genus) reuteri reuteri NO:376 26. Lactobacillus Lactobacillus 100%  100% Lactobacillus 100%  100%Ascusbbr_409(D) SEQ ID 0.50572 (Genus) reuteri reuteri NO: 377 27.Leuconostoc Leuconostoc 99% 100% Leuconostoc 99% 100% Ascusbbr_127 SEQID 0.4955 (Genus) mesenteroides mesenteroides NO: 20 28. LachnospiraceaLachnospiraceae 96% 100% Eubacterium 91% 100% Ascusbbr_14834 SEQ ID0.49531 incertae sedis bacterium hallii NO: 21 (Genus) 29. LactobacillusLactobacillus 99% 100% Lactobacillus 99% 100% Ascusbbr_331885(A) SEQ ID0.49378 (Genus) reuteri reuteri NO: 22 30. Lactobacillus Lactobacillus99% 100% Lactobacillus 99% 100% Ascusbbr_331885(B) SEQ ID 0.49378(Genus) reuteri reuteri NO: 378 31. Lactobacillus Lactobacillus 99% 100%Lactobacillus 99% 100% Ascusbbr_331885(C) SEQ ID 0.49378 (Genus) reuterireuteri NO: 379 32. Anaerofilum Clostridiales 88%  97% Ruthenibacterium88%  98% Ascusbbr_31 SEQ ID 0.48633 (Genus) bacterium lactatiformans NO:23 33. Lachnospiracea Blautia 96% 100% Blautia 96% 100% Ascusbbr_2307SEQ ID 0.48546 incertae sedis hydroge- hydroge- NO: 24 (Genus)notrophica notrophica 34. Lachnospiraceae Clostridium 98% 100%Clostridium 91% 100% Ascusbbr_247(A) SEQ ID 0.48546 Clostridiumsaccharolyticum- clostridioforme NO: 25 Cluster like K10 XIVa (Family +Cluster) 35. Lachnospiraceae Clostridium 98% 100% Clostridium 91% 100%Ascusbbr_247(B) SEQ ID 0.48546 Clostridium saccharolyticum-clostridioforme NO: 380 Cluster like K10 XIVa (Family + Cluster) 36.Microbacterium Pseudo- 95%  99% Pseudo- 99%  79% Ascusbbr_19 SEQ ID0.47772 (Genus) clavibacter sp. clavibacter NO: 26 caeni 37.Verrucosispora Verrucosispora 99% 100% Verrucosispora 97%  98%Ascusbbr_69 SEQ ID 0.47757 (Genus) sp. wenchangensis NO: 27 38.Anaerofilum Faecalibacterium 93% 100% Faecalibacterium 93% 100%Ascusbbr_94 SEQ ID 0.46645 (Genus) prausnitzii prausnitzii NO: 28 39.Clostridium sensu Candidatus 90%  91% Peptoclostridium 89%  91%Ascusbbr_313454 SEQ ID 0.46594 stricto (Genus) Arthromitus sp difficileNO: 29 40. Lactobacillus Lactobacillus 96% 100% Lactobacillus 96% 100%Ascusbbr_351000 SEQ ID 0.46296 (Genus) helveticus helveticus NO: 30 41.Lactobacillus Lactobacillus 99% 100% Lactobacillus 99% 100%Ascusbbr_1436(A) SEQ ID 0.46076 (Genus) salivarius salivarius NO: 31 42.Lactobacillus Lactobacillus 99% 100% Lactobacillus 99% 100%Ascusbbr_1436(B) SEQ ID 0.46076 (Genus) salivarius salivarius NO: 38343. Lachnospiraceae Roseburia 93% 100% Roseburia 93% 100% Ascusbbr_28SEQ ID 0.46028 Clostridium inulinivorans inulinivorans NO: 32 ClusterXIVa (Family + Cluster) 44. Blautia Ruminococcus 94% 100% Ruminococcus94% 100% Ascusbbr_144 SEQ ID 0.45742 (Genus) obeum obeum NO: 33 45.Lactobacillus Lactobacillus 99%  99% Lactobacillus 99%  99%Ascusbbr_42760(A) SEQ ID 0.43682 (Genus) oris oris NO: 34 46.Lactobacillus Lactobacillus 99%  99% Lactobacillus 99%  99%Ascusbbr_42760(B) SEQ ID 0.43682 (Genus) oris oris NO: 384 47.Lactobacillus Lactobacillus 99%  83% Lactobacillus 99%  83%Ascusbbr_134994 SEQ ID 0.434 (Genus) crispatus crispatus NO: 35 48.Lactobacillus Lactobacillus 94%  88% Lactobacillus 94%  88%Ascusbbr_358773 SEQ ID 0.43348 (Genus) reuteri reuteri NO: 36 49.Pseudomonas Pseudomonas 99% 100% Pseudomonas 99% 100% Ascusbbr_2503 SEQID 0.42622 (Genus) chengduensis chengduensis NO: 37 50. SporobacterSyntropho- 86%  82% Clostridium 84%  82% Ascusbbr_312 SEQ ID 0.42622(Genus) monadaceae sphenoides NO: 39 bacterium 51. LactobacillusLactobacillus 99%  83% Lactobacillus 99%  83% Ascusbbr_140914 SEQ ID0.40935 (Genus) crispatus crispatus NO: 40 52. LactobacillusLactobacillus 98%  82% Lactobacillus 98%  82% Ascusbbr_257627 SEQ ID0.40775 (Genus) salivarius salivarius NO: 41 53. LactobacillusLactobacillus 98%  82% Lactobacillus 98%  82% Ascusbbr_310088 SEQ ID0.40576 (Genus) helveticus helveticus NO: 42 54. Lachnospiracea Blautia96% 100% Blautia 96% 100% Ascusbbr_91 SEQ ID 0.40345 incertae sedishydrogeno- hydrogeno- NO:43 (Genus) trophica trophica 55. LactobacillusLactobacillus 97%  83% Lactobacillus 97%  83% Ascusbbr_150100 SEQ ID0.40128 (Genus) crispatus crispatus NO: 44 56. LactobacillusLactobacillus 97% 100% Lactobacillus 97% 100% Ascusbbr_252028 SEQ ID0.3998 (Genus) vaginalis vaginalis NO: 45 57. Peptostrepto- Clostridiumsp. 93% 100% Romboutsia 92% 100% Ascusbbr_2158 SEQ ID 0.39816 coccaceaelituseburensis NO: 46 Clostridium Cluster XI (Family + Cluster) 58.Lactobacillus Lactobacillus 96% 100% Lactobacillus 96% 100% Ascusbbr_373SEQ ID 0.37614 (Genus) crispatus crispatus NO: 47 59. LactobacillusLactobacillus 98%  94% Lactobacillus 98%  94% Ascusbbr_1802 SEQ ID0.37123 (Genus) johnsonii johnsonii NO: 48 60. LactobacillusLactobacillus 96% 100% Lactobacillus 96% 100% Ascusbbr_107 SEQ ID0.37123 (Genus) reuteri reuteri NO: 49 61. Lactobacillus Lactobacillus93%  83% Lactobacillus 93%  83% Ascusbbr_1727 SEQ ID 0.36309 (Genus)crispatus crispatus NO: 50 62. Corynebacterium Corynebacterium 100% 100% Corynebacterium 100%  100% Ascusbbr_226 SEQ ID 0.75897 (Genus)glutamicum glutamicum NO: 338 63. Streptococcus Streptococcus 97%  94%Streptococcus 97%  94% Ascusbbr_17 SEQ ID 0.62924 (Genus) hyovaginalishyovaginalis NO: 339 64. Lactobacillus Lactobacillus 99% 100%Lactobacillus 99% 100% Ascusbbr_14690(A) SEQ ID 0.60061 (Genus) aviariusaviarius NO: 340 65. Lactobacillus Lactobacillus 99% 100% Lactobacillus99% 100% Ascusbbr_14690(B) SEQ ID 0.60061 (Genus) aviarius aviarius NO:371 66. Lactobacillus Lactobacillus 99% 100% Lactobacillus 99% 100%Ascusbbr_14690(C) SEQ ID 0.60061 (Genus) aviarius aviarius NO: 372 67.Corynebacterium Corynebacterium 100%  100% Corynebacterium 100%  100%Ascusbbr_18 SEQ ID 0.58366 (Genus) xerosis xerosis NO: 341 68.Peptostrepto- Romboutsia 98% 100% Romboutsia 98% 100% Ascusbbr_7363 SEQID 0.57242 coccaceae lituseburensis lituseburensis NO: 342 (ClostridiumCluster XI) (Family + Cluster) 69. Corynebacterium Corynebacterium 100% 100% Corynebacterium 100%  100% Ascusbbr_35 SEQ ID 0.49929 (Genus)falsenii falsenii NO: 343 70. Corynebacterium Corynebacterium 98%  97%Corynebacterium 98%  97% Ascusbbr_7779 SEQ ID 0.48127 (Genus)ammoniagenes ammoniagenes NO: 344 71. Lachnospiraceae Desulfoto- 96%100% Clostridium 95% 100% Ascusbbr_10593(A) SEQ ID 0.47763 (Clostridiummaculum sp. sphenoides NO: 345 Cluster XlVa) (Family + Cluster) 72.Lachnospiraceae Desulfoto- 96% 100% Clostridium 95% 100%Ascusbbr_10593(B) SEQ ID 0.47763 (Clostridium maculum sp. sphenoides NO:381 Cluster XlVa) (Family + Cluster) 73. Lachnospiracea Eubacterium sp.98% 100% Eubacterium 93%  98% Ascusbbr_32731(A) SEQ ID 0.47124 incertaesedis fissicatena NO: 346 (Genus) 74. Lachnospiracea Eubacterium sp. 98%100% Eubacterium 93%  98% Ascusbbr_32731(B) SEQ ID 0.47124 incertaesedis fissicatena NO: 382 (Genus) 75. Ruminococcaceae Bacterium 89%  78%Rumini- 86%  79% Ascusbbr_359892 SEQ ID 0.39553 (Clostridium clostridiumNO: 347 Cluster thermocellum III) (Family + Cluster) 76. LactobacillusLactobacillus 100%  100% Lactobacillus 100%  100% Ascusbbr_25721 SEQ ID0.39537 (Genus) pentosus pentosus NO: 348 77. Streptococcus Swine fecal100%  100% Streptococcus 99% 100% Ascusbbr_72076 SEQ ID 0.38425 (Genus)bacterium alactolyticus NO: 349 78. Lachnospiraceae Lachnospiraceae 91% 92% Blautia producta 89%  97% Ascusbbr_6097 SEQ ID 0.37484 (Clostridiumbacterium NO: 350 Cluster XlVa) (Family + Cluster) 79. LactobacillusLactobacillus 100%  100% Lactobacillus 100%  100% Ascusbbr_265(A) SEQ ID0.37167 (Genus) helveticus helveticus NO: 351 80. LactobacillusLactobacillus 100%  100% Lactobacillus 100%  100% Ascusbbr_265(B) SEQ ID0.37167 (Genus) helveticus helveticus NO: 385 81. Paracoccus Paracoccus99%  99% Paracoccus 99%  99% Ascusbbr_323376 SEQ ID 0.36852 (Genus)alcaliphilus alcaliphilus NO: 352 82. Cellulosilyticum Ruminococcus sp.97% 100% Hydrogenoan 84%  98% Ascusbbr_36257 SEQ ID 0.36078 (Genus)aerobacterium NO: 353 saccharovorans 83. Blautia Blautia 89% 100%Blautia 89% 100% Ascusbbr_6957 SEQ ID 0.35528 (Genus) gluceraseaglucerasea NO: 354 84. Corynebacterium Corynebacterium 99% 100%Corynebacterium 99% 100% Ascusbbr_38 SEQ ID 0.35488 (Genus) flavescensflavescens NO: 355 85. Lachnospiracea Eubacteriaceae 98%  92%Coprococcus 87%  99% Ascusbbr_13398 SEQ ID 0.34774 incertae sedisbacterium catus NO: 356 (Genus) 86. Corynebacterium Corynebacterium100%  100% Corynebacterium 100%  100% Ascusbbr_57 SEQ ID 0.34405 (Genus)callunae callunae NO: 357 87. Corynebacterium Corynebacterium 99% 100%Corynebacterium 99% 100% Ascusbbr_285160 SEQ ID 0.32892 (Genus)stationis stationis NO: 358 88. Ruminococcus Clostridium sp. 92%  96%Blautia producta 91%  96% Ascusbbr_37385 SEQ ID 0.3236 (Genus) NO: 35989. Lactobacillus Lactobacillus 92% 100% Lactobacillus 92% 100%Ascusbbr_118124 SEQ ID 0.31115 (Genus) intestinalis intestinalis NO: 36090. Roseburia Bacterium 92%  99% Frisingicoccus 91% 100% Ascusbbr_32592SEQ ID 0.29912 (Genus) AC2012 caecimuris NO: 361 91. LachnospiraceaeClostridium sp. 90%  99% Eubacterium 92%  91% Ascusbbr_110856 SEQ ID0.29418 (Clostridium ventriosum NO: 362 Cluster XlVa) (Family + Cluster)92. Lachnospiraceae Clostridiales 99%  99% Clostridium 97%  99%Ascusbbr_185064 SEQ ID 0.21604 (Clostridium bacterium lactatifermentansNO: 363 Cluster XlVb) (Family + Cluster) 93. Clostridium sensuClostridium sp. 99% 100% Clostridium 99%  99% Ascusbbr _3315 SEQ ID0.20534 stricto (Genus) thermobutyricum NO: 364 94. Bacteroides doreiBacteroides dorei 100%  100% Bacteroides dorei 100%  100% Ascusbbr_578SEQ ID 0.74887 NO: 365 95. Lactobacillus Lactobacillus 95% 100%Lactobacillus 95% 100% Ascusbbr_21169 SEQ ID 0.47787 (Genus) reuterireuteri NO: 366 96. Lactobacillus Lactobacillus 96%  98% Lactobacillus96%  98% Ascusbbr_110856 SEQ ID 0.39178 (Genus) reuteri reuteri NO: 36797. Lactobacillus Lactobacillus 100%  100% Lactobacillus 100%  100%Ascusbbr_830 SEQ ID 0.33782 (Genus) saerimneri saerimneri NO: 368 98.Clostridium Clostridium 99% 100% Clostridium 99% 100% Ascusbbr_2676 SEQID 0.40777 (Genus) beijerinckii beijerinckii NO: 386 99. ClostridiumClostridium 100%   99% Clostridium 100%   99% Ascusbbr_105932 SEQ ID0.47763 (Genus) saccharolyticum saccharolyticum NO: 387 100. NectriaceaeFusarium 100%  100% Fusarium 100%  100% Ascusfbr_15 SEQ ID 0.42622(Family) annulatum annulatum NO: 51 101. Filobasidium Uncultured 100% 100% Cryptococcus 100%  100% Ascusfbr _131 SEQ ID 0.42622 floriformefungus magnus NO: 52 (Genus + species) 102. Gibberella zeae Fusarium100%  100% Fusarium 100%  100% Ascusfbr _26 SEQ ID 0.36913 (Genus +species) asiaticum asiaticum NO: 53 103. Alatospora Uncultured 83%  81%Gymnoascus 83%  81% Ascusfbr _2616 SEQ ID 0.33927 (Genus) Gymnoascusreesii NO: 54 104. Hypocreaceae Geotrichum sp. 100%  100% Geotrichum100%  100% Ascusfbr _12 SEQ ID 0.32217 (Family) candidum NO: 55 105.Pichia Pichia 100%  100% Pichia 100%  100% Ascusfbr _53 SEQ ID 0.30645fermentans fermentans fermentans NO: 56 (Genus + species) 106. CandidaCandida 99% 100% Candida 99% 100% Ascusfbr _1379 SEQ ID 0.28513railenensis railenensis railenensis NO: 57 (Genus + species) 107.Hypocreaceae Uncultured 100%  100% Geotrichum 100%  100% Ascusfbr _122SEQ ID 0.25801 (Family) fungus candidum NO: 58

In some embodiments, the isolated microbial strains of the presentdisclosure further encompass mutants thereof. In some embodiments, thepresent disclosure further contemplates microbial strains having all ofthe identifying characteristics of the presently disclosed microbialstrains.

TABLE 2 Microbial Deposits Corresponding to the Microbes of Table 1 Se-Se- quence quence Identi- Identi- fier for fier for Predicted Taxa Asso-Predicted Taxa Asso- of Isolated Strain ciated of Isolated Strain ciatedMicrobes Designation Marker Deposit # Microbes Designation MarkerDeposit # Lactobacillus Ascusbbr_4729 SEQ ID PATENT201703004Peptostrepto- Ascusbbr_2158 SEQ ID PTA-124039 (Genus) NO: 1 coccaceaeNO: 46 Clostridium Cluster XI (Family + Cluster) LachnospiraceaeAscusbbr_339 SEQ ID PTA-124016, Lactobacillus Ascusbbr_373 SEQ IDClostridium NO: 2 PTA-124039 (Genus) NO: 47 Cluster XIVa (Family +Cluster) Lactobacillus Ascusbbr_5796(A) SEQ ID PATENT201703001,Lactobacillus Ascusbbr_1802 SEQ ID (Genus) NO: 3 PATENT201703003,(Genus) NO: 48 PATENT201703004, B-67267 Lactobacillus Ascusbbr_5796(B)SEQ ID PTA-124039 (Genus) NO: 369 Lactobacillus Ascusbbr_5796(C) SEQ IDPATENT201703002 (Genus) NO: 370 Lactobacillus Ascusbbr_38717(A) SEQ IDPATENT201703002, Lactobacillus Ascusbbr_107 SEQ ID PATENT201703002(Genus) NO: 4 PATENT201703003, (Genus) NO: 49 PATENT201703004, B-67268Lactobacillus Ascusbbr_38717(B) SEQ ID PATENT201703001 (Genus) NO: 373Lactobacillus Ascusbbr_170211 SEQ ID PATENT201703002 LactobacillusAscusbbr_1727 SEQ ID (Genus) NO: 5 (Genus) NO: 50 LactobacillusAscusbbr_1686 SEQ ID PTA-124016, Coryne- Ascusbbr_226 SEQ IDPATENT201703003 (Genus) NO: 6 PTA-124039, bacterium NO: PATENT201703001,(Genus) 338 PATENT201703002, PATENT201703003, PATENT201703004, B-67270Faecali- Ascusbbr_1789 SEQ ID PTA-124016, Streptococcus Ascusbbr_17 SEQID PATENT201703002, bacterium NO: 7 PTA-124039, (Genus) NO:PATENT201703003, (Genus) PATENT201703001 339 PATENT201703004Lactobacillus Ascusbbr_3820 SEQ ID PTA-124039, LactobacillusAscusbbr_14690(A) SEQ ID PTA-124039, (Genus) NO: 8 PATENT201703002(Genus) NO: PATENT201703001, 340 PATENT201703002, PATENT201703003,PATENT201703004 Lactobacillus Ascusbbr_14690(B) SEQ ID PTA-124016,(Genus) NO: 371 Lactobacillus Ascusbbr_14690(C) SEQ ID PATENT201703004(Genus) NO: 372 Hydro- Ascusbbr_173 SEQ ID PTA-124016, Coryne-Ascusbbr_18 SEQ ID PATENT201703003 genoanaero NO: 9 PTA-124039 bacteriumNO: bacterium (Genus) 341 (Genus) Peptostrepto- Ascusbbr_3089 SEQ IDPTA-124016 Peptostrepto- Ascusbbr_7363 SEQ ID PTA-124016 coccaceae NO:10 coccaceae NO: Clostridium (Clostridium 342 Cluster XI Cluster XI)(Family + (Family + Cluster) Cluster) Acrocar- Ascusbbr_167 SEQ IDCoryne- Ascusbbr_35 SEQ ID PATENT201703002, pospora NO: 11 bacterium NO:PATENT201703003, (Genus) (Genus) 343 PATENT201703004 LactobacillusAscusbbr_301568 SEQ ID Coryne- Ascusbbr_7779 SEQ ID PATENT201703002,(Genus) NO: 12 bacterium NO: PATENT201703003, (Genus) 344PATENT201703004 Bacillus Ascusbbr_33(A) SEQ ID PATENT201703002, Lachno-Ascusbbr_10593(A) SEQ ID PTA-124039, (Genus) NO: 13 PATENT201703003,spiraceae NO: PATENT201703001, B-67266 (Clostridium 345 PATENT201703002,Cluster XlVa) PATENT201703003 (Family + Cluster) Bacillus Ascusbbr_33(B)SEQ ID PATENT201703001, Lachno- Ascusbbr_10593(B) SEQ ID PTA-124016,(Genus) NO: spiraceae NO: 374 (Clostridium 381 Cluster XlVa) (Family +Cluster) Lactobacillus Ascusbbr_25200 SEQ ID PATENT201703001, Lachno-Ascusbbr_32731(A) SEQ ID PTA-124016, (Genus) NO: 14 PATENT201703002,spiracea NO: PATENT201703001, PATENT201703003, incertae sedis 346PATENT201703002 PATENT201703004 (Genus) Lachno- Ascusbbr_32731(B) SEQ IDPTA-124039 spiracea NO: incertae sedis 382 (Genus) Subdoli- Ascusbbr_84SEQ ID PTA-124039, Rumino- Ascusbbr_359892 SEQ ID PTA-124039 granulumNO: 15 PATENT201703003 coccaceae NO: (Genus) (Clostridium 347 ClusterIII) (Family + Cluster) Subdoli- Ascusbbr_136 SEQ ID PTA-124016Lactobacillus Ascusbbr_25721 SEQ ID granulum NO: 16 (Genus) NO: (Genus)348 Lachno- Ascusbbr_128 SEQ ID PATENT201703004 StreptococcusAscusbbr_72076 SEQ ID PTA-124039, spiraceae NO: 17 (Genus) NO:PATENT201703001, Clostridium 349 PATENT201703002, Cluster XIVaPATENT201703003, (Family + PATENT201703004 Cluster) LactobacillusAscusbbr_322104 SEQ ID PATENT201703001 Lachno- Ascusbbr_6097 SEQ IDPTA-124016, (Genus) NO: 18 spiraceae NO: PTA-124039, (Clostridium 350PATENT201703002, Cluster XlVa) PATENT201703003, (Family +PATENT201703004 Cluster) Lactobacillus Ascusbbr_409(A) SEQ IDPTA-124039, Lactobacillus Ascusbbr_265 (A) SEQ ID PATENT201703001,(Genus) NO: 19 PATENT201703002, (Genus) NO: PATENT201703002PATENT201703003, 351 Lactobacillus Ascusbbr_409(B) SEQ IDPATENT201703004 PATENT201703004 (Genus) NO: 375 LactobacillusAscusbbr_409(C) SEQ ID PATENT201703001 (Genus) NO: 376 LactobacillusAscusbbr_409(D) SEQ ID PATENT201703001 Lactobacillus Ascusbbr_265 (B)SEQ ID PATENT201703001, (Genus) NO: (Genus) NO: PATENT201703002, 377 385PATENT201703004 Leuconostoc Ascusbbr_127 SEQ ID B-67265 ParacoccusAscusbbr_323376 SEQ ID PATENT201703002 (Genus) NO: 20 (Genus) NO: 352Lachno- Ascusbbr_14834 SEQ ID PTA-124016, Cellulo- Ascusbbr_36257 SEQ IDPTA-124039 spiracea NO: 21 PTA-124039, silyticum NO: incertae sedisPATENT201703001, (Genus) 353 (Genus) PATENT201703002, PATENT201703003Lactobacillus Ascusbbr_331885(A) SEQ ID PTA-124039, BlautiaAscusbbr_6957 SEQ ID PTA-124039, (Genus) NO: 22 PATENT201703002, (Genus)NO: PATENT201703002, PATENT201703003, 354 PATENT201703003 B-67269Lactobacillus Ascusbbr_331885(B) SEQ ID PATENT201703001 (Genus) NO: 378Lactobacillus Ascusbbr_331885(C) SEQ ID PATENT201703004 (Genus) NO: 379Anaerofilum Ascusbbr_31 SEQ ID PTA-124039, Coryne- Ascusbbr_38 SEQ IDPATENT201703003 (Genus) NO: 23 PATENT201703002 bacterium NO: (Genus) 355Lachno- Ascusbbr_2307 SEQ ID PTA-124039 Lachno- Ascusbbr_13398 SEQ IDPTA-124039, spiracea NO: 24 spiracea NO: PATENT201703003 incertae sedisincertae sedis 356 (Genus) (Genus) Lachno- Ascusbbr_247(A) SEQ IDPTA-124039, Coryne- Ascusbbr_57 SEQ ID PATENT201703003 spiraceae NO: 25PATENT201703004 bacterium NO: Clostridium (Genus) 357 Cluster XIVa(Family + Cluster) Lachno- Ascusbbr_247(B) SEQ ID PTA-124016 spiraceaeNO: Clostridium 380 Cluster XIVa (Family + Cluster) MicrobacteriumAscusbbr_19 SEQ ID PATENT201703001, Coryne- Ascusbbr_285160 SEQ IDPTA-124039, (Genus) NO: 26 B-67264 bacterium NO: PATENT201703001,(Genus) 358 PATENT201703002, PATENT201703003, PATENT201703004Verrucosispora Ascusbbr_69 SEQ ID Ruminococcus Ascusbbr_37385 SEQ ID(Genus) NO: 27 (Genus) NO: 359 Anaerofilum Ascusbbr_94 SEQ IDPATENT201703001, Lactobacillus Ascusbbr_118124 SEQ ID PATENT201703001(Genus) NO: 28 PATENT201703004 (Genus) NO: 360 ClostridiumAscusbbr_313454 SEQ ID PATENT201703003, Roseburia Ascusbbr_32592 SEQ IDPATENT201703002 sensu stricto NO: 29 PATENT201703004 (Genus) NO: (Genus)361 Lactobacillus Ascusbbr_351000 SEQ ID Lachno- Ascusbbr_110856 SEQ IDPATENT201703004 (Genus) NO: 30 spiraceae NO: (Clostridium 362 ClusterXlVa) (Family + Cluster) Lactobacillus Ascusbbr_1436(A) SEQ IDPTA-124039, Lachno- Ascusbbr_185064 SEQ ID PTA-124039, (Genus) NO: 31PATENT201703001, spiraceae NO: PATENT201703001, PATENT201703002,(Clostridium 363 PATENT201703002 PATENT201703003, Cluster XlVb)PATENT201703004 (Family + Cluster) Lactobacillus Ascusbbr_1436(B) SEQ IDPTA-124016 (Genus) NO: 383 Lachno- Ascusbbr_28 SEQ ID PTA-124016,Clostridium Ascusbbr_3315 SEQ ID spiraceae NO: 32 PTA-124039, sensustricto NO: Clostridium PATENT201703002 (Genus) 364 Cluster XIVa(Family + Cluster) Blautia (Genus) Ascusbbr_144 SEQ ID PTA-124039,Bacteroides Ascusbbr_578 SEQ ID PTA-124039 NO: 33 PATENT201703002 doreiNO: 365 Lactobacillus Ascusbbr_42760(A) SEQ ID PTA-124039, LactobacillusAscusbbr_21169 SEQ ID PATENT201703001 (Genus) NO: 34 PATENT201703002,(Genus) NO: PATENT201703003, 366 PATENT201703004 LactobacillusAscusbbr_42760(B) SEQ ID PATENT201703001 (Genus) NO: 384 LactobacillusAscusbbr_134994 SEQ ID Lactobacillus Ascusbbr_48584 SEQ IDPATENT201703004 (Genus) NO: 35 (Genus) NO: 367 LactobacillusAscusbbr_358773 SEQ ID Nectriaceae Ascusfbr_15 SEQ ID (Genus) NO: 36(Family) NO: 51 Pseudomonas Ascusbbr_2503 SEQ ID PATENT201703001Filobasidium Ascusfbr_131 SEQ ID (Genus) NO: 37 floriforme NO: 52(Genus + species) Sporobacter Ascusbbr_312 SEQ ID PATENT201703002Gibberella Ascusfbr_26 SEQ ID (Genus) NO: 39 zeae (Genus NO: 53 +species) Lactobacillus Ascusbbr_140914 SEQ ID Alatospora Ascusfbr_2616SEQ ID (Genus) NO: 40 (Genus) NO: 54 Lactobacillus Ascusbbr_257627 SEQID Hypocreaceae Ascusfbr_12 SEQ ID (Genus) NO: 41 (Family) NO: 55Lactobacillus Ascusbbr_310088 SEQ ID Pichia Ascusfbr_53 SEQ ID (Genus)NO: 42 fermentans NO: 56 (Genus + species) Lachno- Ascusbbr_91 SEQ IDPTA-124016, Candida Ascusfbr_1379 SEQ ID spiracea NO: 43 PTA-124039,railenensis NO: 57 incertae sedis PATENT201703002, (Genus + (Genus)PATENT201703003 species) Lactobacillus Ascusbbr_150100 SEQ IDHypocreaceae Ascusfbr_122 SEQ ID (Genus) NO: 44 (Family) NO: 58Lactobacillus Ascusbbr_252028 SEQ ID Lactobacillus Ascusbbr_830 SEQ IDPTA-124039 (Genus) NO: 45 (Genus) NO: 368 Clostridium Ascusbbr_2676 SEQID PATENT201703003 Clostridium Ascusbbr_105932 SEQ ID PATENT201703001beijerinckii NO: PATENT201703004 saccharo- NO: PATENT201703002 (Genus +386 lyticum 387 species) (Genus + species)

TABLE 3 Bacteria of the present disclosure. Sequence Sequence Identifierfor Identifier for Predicted Closest Taxa of Strain Associated PredictedClosest Taxa of Strain Associated Isolated Microbes Designation MarkerIsolated Microbes Designation Marker 1. Clostridium XIVb (Cluster)Ascusbbr_6 SEQ ID Actinomyces (Genus) Ascusbbr_2226 SEQ ID NO: 59 NO:199 2. Gemmiger (Genus) Ascusbbr_113 SEQ ID Succiniclasticum (Genus)Ascusbbr_2227 SEQ ID NO: 60 NO: 200 3. Lactobacillus (Genus)Ascusbbr_116 SEQ ID Beijerinckia (Genus) Ascusbbr_2229 SEQ ID NO: 61 NO:201 4. Clostridium XI (Cluster) Ascusbbr_129 SEQ ID Bosea (Genus)Ascusbbr_2235 SEQ ID NO: 62 NO: 202 5. Jeotgalicoccus (Genus)Ascusbbr_265 SEQ ID Sporobacter (Genus) Ascusbbr_2237 SEQ ID NO: 63 NO:203 6. Lactobacillus (Genus) Ascusbbr_275 SEQ ID Facklamia (Genus)Ascusbbr_2251 SEQ ID NO: 64 NO: 204 7. Lactobacillus (Genus)Ascusbbr_343 SEQ ID Acinetobacter (Genus) Ascusbbr_2266 SEQ ID NO: 65NO: 205 8. Lactobacillus (Genus) Ascusbbr_363 SEQ ID Brevundimonas(Genus) Ascusbbr_2284 SEQ ID NO: 66 NO: 206 9. Jeotgalicoccus (Genus)Ascusbbr_399 SEQ ID Ochrobactrum (Genus) Ascusbbr_2285 SEQ ID NO: 67 NO:207 10. Lactobacillus (Genus) Ascusbbr_444 SEQ ID Alcaligenes (Genus)Ascusbbr_2290 SEQ ID NO: 68 NO: 208 11. Jeotgalicoccus (Genus)Ascusbbr_498 SEQ ID Pseudochrobactrum (Genus) Ascusbbr_2291 SEQ ID NO:69 NO: 209 12. Lactobacillus (Genus) Ascusbbr_542 SEQ ID Jeotgalicoccus(Genus) Ascusbbr_2292 SEQ ID NO: 70 NO: 210 13. Lactobacillus (Genus)Ascusbbr_561 SEQ ID Jeotgalicoccus (Genus) Ascusbbr_2293 SEQ ID NO: 71NO: 211 14. Lactobacillus (Genus) Ascusbbr_570 SEQ ID Acinetobacter(Genus) Ascusbbr_2294 SEQ ID NO: 72 NO: 212 15. Corynebacterium (Genus)Ascusbbr_616 SEQ ID Sphingobacterium (Genus) Ascusbbr_2295 SEQ ID NO: 73NO: 213 16. Microbacterium (Genus) Ascusbbr_620 SEQ ID Lachnospiracea(Genus) Ascusbbr_2301 SEQ ID NO: 74 NO: 214 17. Jeotgalicoccus (Genus)Ascusbbr_690 SEQ ID Azospirillum (Genus) Ascusbbr_2302 SEQ ID NO: 75 NO:215 18. Jeotgalicoccus (Genus) Ascusbbr_705 SEQ ID Lactobacillus (Genus)Ascusbbr_2313 SEQ ID NO: 76 NO: 216 19. Glycomyces (Genus) Ascusbbr_793SEQ ID Clavibacter (Genus) Ascusbbr_2320 SEQ ID NO: 77 NO: 217 20.Streptomyces (Genus) Ascusbbr_795 SEQ ID Clostridium XIVa (Cluster)Ascusbbr_2324 SEQ ID NO: 78 NO: 218 21. Saccharopolyspora (Genus)Ascusbbr_796 SEQ ID Clostridium XIVa (Cluster) Ascusbbr_2325 SEQ ID NO:79 NO: 219 22. Brevibacterium (Genus) Ascusbbr_803 SEQ ID Lactobacillus(Genus) Ascusbbr_2328 SEQ ID NO: 80 NO: 220 23. Microbacterium (Genus)Ascusbbr_804 SEQ ID Clostridium XIVa (Cluster) Ascusbbr_2331 SEQ ID NO:81 NO: 221 24. Acinetobacter (Genus) Ascusbbr_840 SEQ ID Bacillus(Genus) Ascusbbr_2337 SEQ ID NO: 82 NO: 222 25. Lactococcus (Genus)Ascusbbr_846 SEQ ID Methanoplanus (Genus) Ascusbbr_2354 SEQ ID NO: 83NO: 223 26. Cloacibacterium (Genus) Ascusbbr_867 SEQ ID Mogibacterium(Genus) Ascusbbr_2361 SEQ ID NO: 84 NO: 224 27. Mycobacterium (Genus)Ascusbbr_929 SEQ ID Brachybacterium (Genus) Ascusbbr_2368 SEQ ID NO: 85NO: 225 28. Leucobacter (Genus) Ascusbbr_944 SEQ ID Facklamia (Genus)Ascusbbr_2376 SEQ ID NO: 86 NO: 226 29. Lactobacillus (Genus)Ascusbbr_950 SEQ ID Clostridium XIVa (Cluster) Ascusbbr_2378 SEQ ID NO:87 NO: 227 30. Rothia (Genus) Ascusbbr_951 SEQ ID Clostridium XIVa(Cluster) Ascusbbr_2380 SEQ ID NO: 88 NO: 228 31. Lactobacillus (Genus)Ascusbbr_996 SEQ ID Syntrophomonas (Genus) Ascusbbr_2383 SEQ ID NO: 89NO: 229 32. Clavibacter (Genus) Ascusbbr_1005 SEQ ID Beijerinckia(Genus) Ascusbbr_2386 SEQ ID NO: 90 NO: 230 33. Hydrogenoanaerobacterium(Genus) Ascusbbr_1029 SEQ ID Lactobacillus (Genus) Ascusbbr_2390 SEQ IDNO: 91 NO: 231 34. Howardella (Genus) Ascusbbr_1036 SEQ ID Lactobacillus(Genus) Ascusbbr_2391 SEQ ID NO: 92 NO: 232 35. Clostridium (Genus)Ascusbbr_1069 SEQ ID Lactobacillus (Genus) Ascusbbr_2395 SEQ ID NO: 93NO: 233 36. Ascusbbr_1128 SEQ ID Erysipelotrichaceae (Family)Ascusbbr_2397 SEQ ID NO: 94 NO: 234 37. Hydrogenoanaerobacterium (Genus)Ascusbbr_1139 SEQ ID Rummeliibacillus (Genus) Ascusbbr_2399 SEQ ID NO:95 NO: 235 38. Papillibacter (Genus) Ascusbbr_1169 SEQ ID Acinetobacter(Genus) Ascusbbr_2402 SEQ ID NO: 96 NO: 236 39. Butyricicoccus (Genus)Ascusbbr_1185 SEQ ID Lactococcus (Genus) Ascusbbr_2403 SEQ ID NO: 97 NO:237 40. Eubacterium (Genus) Ascusbbr_1245 SEQ ID Propionibacterium(Genus) Ascusbbr_2412 SEQ ID NO: 98 NO: 238 41. Turicibacter (Genus)Ascusbbr_1258 SEQ ID Clostridium (Genus) Ascusbbr_2413 SEQ ID NO: 99 NO:239 42. Lactobacillus (Genus) Ascusbbr_1264 SEQ ID Clostridium XIVa(Cluster) Ascusbbr_2416 SEQ ID NO: 100 NO: 240 43. Asaccharobacter(Genus) Ascusbbr_1332 SEQ ID Rummeliibacillus (Genus) Ascusbbr_2419 SEQID NO: 101 NO: 241 44. Faecalibacterium (Genus) Ascusbbr_1360 SEQ IDRalstonia (Genus) Ascusbbr_2420 SEQ ID NO: 102 NO: 242 45. ClostridiumXIVa (Cluster) Ascusbbr_1363 SEQ ID Brachybacterium (Genus)Ascusbbr_2421 SEQ ID NO: 103 NO: 243 46. Clostridium XIVa (Cluster)Ascusbbr_1422 SEQ ID Ruminobacter (Genus) Ascusbbr_2423 SEQ ID NO: 104NO: 244 47. Clostridium IV (Cluster) Ascusbbr_1424 SEQ ID Glycomyces(Genus) Ascusbbr_2427 SEQ ID NO: 105 NO: 245 48. Clostridium XIVb(Cluster) Ascusbbr_1433 SEQ ID Psychrobacter (Genus) Ascusbbr_2428 SEQID NO: 106 NO: 246 49. Butyricicoccus (Genus) Ascusbbr_1456 SEQ IDYaniella (Genus) Ascusbbr_2429 SEQ ID NO: 107 NO: 247 50. Sporobacter(Genus) Ascusbbr_1485 SEQ ID Clostridium IV (Cluster) Ascusbbr_2431 SEQID NO: 108 NO: 248 51. Butyricicoccus (Genus) Ascusbbr_1488 SEQ IDClostridium (Genus) Ascusbbr_2434 SEQ ID NO: 109 NO: 249 52.Hydrogenoanaerobacterium (Genus) Ascusbbr_1490 SEQ ID Clostridium XIVa(Cluster) Ascusbbr_2435 SEQ ID NO: 110 NO: 250 53. Anaerofilum (Genus)Ascusbbr_1493 SEQ ID Clostridium (Genus) Ascusbbr_2436 SEQ ID NO: 111NO: 251 54. Clostridium XIVa (Cluster) Ascusbbr_1536 SEQ ID ClostridiumXIVa (Cluster) Ascusbbr_2437 SEQ ID NO: 112 NO: 252 55. Clostridium XIVa(Cluster) Ascusbbr_1541 SEQ ID Cohnella (Genus) Ascusbbr_2438 SEQ ID NO:113 NO: 253 56. Lachnospiracea (Family) Ascusbbr_1572 SEQ IDChthonomonas (Genus) Ascusbbr_2441 SEQ ID NO: 114 NO: 254 57.Lachnospiracea (Family) Ascusbbr_1592 SEQ ID Streptophyta (UnrankedAscusbbr_2445 SEQ ID NO: 115 Clade) NO: 255 58. Butyricicoccus (Genus)Ascusbbr_1611 SEQ ID Acinetobacter (Genus) Ascusbbr_2452 SEQ ID NO: 116NO: 256 59. Pediococcus (Genus) Ascusbbr_1614 SEQ ID Clostridium XIVbAscusbbr_2456 SEQ ID NO: 117 NO: 257 60. Acetanaerobacterium (Genus)Ascusbbr_1616 SEQ ID Neisseria Ascusbbr_2465 SEQ ID NO: 118 NO: 258 61.Hydrogenoanaerobacterium (Genus) Ascusbbr_1623 SEQ ID ButyricicoccusAscusbbr_2471 SEQ ID NO: 119 NO: 259 62. Butyricicoccus (Genus)Ascusbbr_1625 SEQ ID Sporobacter Ascusbbr_2472 SEQ ID NO: 120 NO: 26063. Lachnospiracea (Family) Ascusbbr_1632 SEQ ID SporobacterAscusbbr_2476 SEQ ID NO: 121 NO: 261 64. Erysipelotrichaceae (Family)Ascusbbr_1634 SEQ ID Syntrophomonas Ascusbbr_2477 SEQ ID NO: 122 NO: 26265. Lachnospiracea (Family) Ascusbbr_1635 SEQ ID DesulfotomaculumAscusbbr_2478 SEQ ID NO: 123 NO: 263 66. Butyricicoccus (Genus)Ascusbbr_1646 SEQ ID Streptophyta Ascusbbr_2482 SEQ ID NO: 124 NO: 26467. Butyricicoccus (Genus) Ascusbbr_1669 SEQ ID AcetomicrobiumAscusbbr_2489 SEQ ID NO: 125 NO: 265 68. Butyricicoccus (Genus)Ascusbbr_1670 SEQ ID Acinetobacter Ascusbbr_2492 SEQ ID NO: 126 NO: 26669. Butyricicoccus (Genus) Ascusbbr_1674 SEQ ID ErysipelotrichaceaeAscusbbr_2493 SEQ ID NO: 127 NO: 267 70. Butyricicoccus (Genus)Ascusbbr_1678 SEQ ID Jeotgalicoccus Ascusbbr_2496 SEQ ID NO: 128 NO: 26871. Lachnospiracea (Family) Ascusbbr_1679 SEQ ID SelenomonasAscusbbr_2497 SEQ ID NO: 129 NO: 269 72. Howardella (Genu) Ascusbbr_1684SEQ ID Howardella Ascusbbr_2498 SEQ ID NO: 130 NO: 270 73.Lachnospiracea (Family) Ascusbbr_1685 SEQ ID Clostridium XIVaAscusbbr_2500 SEQ ID NO: 131 NO: 271 74. Clavibacter (Genus)Ascusbbr_1694 SEQ ID Lachnospiracea Ascusbbr_2501 SEQ ID NO: 132 NO: 27275. Butyricicoccus (Genus) Ascusbbr_1695 SEQ ID LachnospiraceaAscusbbr_2504 SEQ ID NO: 133 NO: 273 76. Hydrogenoanaerobacterium(Genus) Ascusbbr_1715 SEQ ID Clostridium XIVa Ascusbbr_2506 SEQ ID NO:134 NO: 274 77. Spiroplasma (Genus) Ascusbbr_1720 SEQ ID LachnospiraceaAscusbbr_2508 SEQ ID NO: 135 NO: 275 78. Clostridium XIVa (Cluster)Ascusbbr_1722 SEQ ID Bacillus Ascusbbr_2509 SEQ ID NO: 136 NO: 276 79.Jeotgalicoccus (Genus) Ascusbbr_1723 SEQ ID Paenibacillus Ascusbbr_2510SEQ ID NO: 137 NO: 277 80. Syntrophomonas (Genus) Ascusbbr_1743 SEQ IDEubacterium Ascusbbr_2511 SEQ ID NO: 138 NO: 278 81. Clostridium IV(Cluster) Ascusbbr_1746 SEQ ID Amphibacillus Ascusbbr_2512 SEQ ID NO:139 NO: 279 82. Lachnospiracea (Family) Ascusbbr_1748 SEQ IDStaphylococcus Ascusbbr_2513 SEQ ID NO: 140 NO: 280 83.Hydrogenoanaerobacterium (Genus) Ascusbbr_1753 SEQ ID PaenibacillusAscusbbr_2514 SEQ ID NO: 141 NO: 281 84. Oscillibacter (Genus)Ascusbbr_1756 SEQ ID Clostridium IV Ascusbbr_2515 SEQ ID NO: 142 NO: 28285. Clostridium IV (Cluster) Ascusbbr_1785 SEQ ID PrevotellaAscusbbr_2516 SEQ ID NO: 143 NO: 283 86. Sporobacter (Genus)Ascusbbr_1812 SEQ ID Barnesiella Ascusbbr_2518 SEQ ID NO: 144 NO: 28487. Pediococcus (Genus) Ascusbbr_1821 SEQ ID Clostridium XIVaAscusbbr_2519 SEQ ID NO: 145 NO: 285 88. Sporobacter (Genus)Ascusbbr_1824 SEQ ID Clostridium XIVa Ascusbbr_2520 SEQ ID NO: 146 NO:286 89. Bacillus (Genus) Ascusbbr_1866 SEQ ID Sharpea Ascusbbr_2521 SEQID NO: 147 NO: 287 90. Cellulomonas (Genus) Ascusbbr_1882 SEQ IDLachnospiracea Ascusbbr_2522 SEQ ID NO: 148 NO: 288 91. Syntrophomonas(Genus) Ascusbbr_1887 SEQ ID Leucobacter Ascusbbr_2523 SEQ ID NO: 149NO: 289 92. Cryptanaerobacter (Genus) Ascusbbr_1928 SEQ ID LactonifactorAscusbbr_2524 SEQ ID NO: 150 NO: 290 93. Sporobacter (Genus)Ascusbbr_1932 SEQ ID Lachnospiracea Ascusbbr_2525 SEQ ID NO: 151 NO: 29194. Hydrogenoanaerobacterium (Genus) Ascusbbr_1933 SEQ IDSucciniclasticum Ascusbbr_2526 SEQ ID NO: 152 NO: 292 95. Clostridium IV(Cluster) Ascusbbr_1937 SEQ ID Acidovorax Ascusbbr_2528 SEQ ID NO: 153NO: 293 96. Hydrogenoanaerobacterium (Genus) Ascusbbr_1953 SEQ IDAcinetobacter Ascusbbr_2530 SEQ ID NO: 154 NO: 294 97. Spiroplasma(Genus) Ascusbbr_1955 SEQ ID Comamonas Ascusbbr_2531 SEQ ID NO: 155 NO:295 98. Erysipelotrichaceae (Family) Ascusbbr_1956 SEQ ID PrevotellaAscusbbr_2533 SEQ ID NO: 156 NO: 296 99. Pseudoflavonifractor (Genus)Ascusbbr_1957 SEQ ID Clostridium IV Ascusbbr_2534 SEQ ID NO: 157 NO: 297100. Clostridium XIVa (Cluster) Ascusbbr_1967 SEQ ID ClostridiumAscusbbr_2535 SEQ ID NO: 158 NO: 298 101. Mogibacterium (Genus)Ascusbbr_1969 SEQ ID Succiniclasticum Ascusbbr_2536 SEQ ID NO: 159 NO:299 102. Clostridium (Genus) Ascusbbr_1973 SEQ ID LachnospiraceaAscusbbr_2538 SEQ ID NO: 160 NO: 300 103. Clostridium IV (Cluster)Ascusbbr_2020 SEQ ID Pedobacter Ascusbbr_2539 SEQ ID NO: 161 NO: 301104. Citrobacter (Genus) Ascusbbr_2023 SEQ ID Clostridium XIIAscusbbr_2540 SEQ ID NO: 162 NO: 302 105. Hydrogenoanaerobacterium(Genus) Ascusbbr_2033 SEQ ID Flavobacterium Ascusbbr_2544 SEQ ID NO: 163NO: 303 106. Clostridium XIVa (Cluster) Ascusbbr_2047 SEQ ID ClostridiumAscusbbr_2545 SEQ ID NO: 164 NO: 304 107. Clostridium XIVa (Cluster)Ascusbbr_2049 SEQ ID Alkaliphilus Ascusbbr_2547 SEQ ID NO: 165 NO: 305108. Clostridium (Genus) Ascusbbr_2057 SEQ ID Arthrobacter Ascusbbr_2548SEQ ID NO: 166 NO: 306 109. Erysipelotrichaceae (Family) Ascusbbr_2069SEQ ID Flavobacterium Ascusbbr_2549 SEQ ID NO: 167 NO: 307 110.Clostridium XIVb (Cluster) Ascusbbr_2073 SEQ ID Roseburia Ascusbbr_2550SEQ ID NO: 168 NO: 308 111. Clostridium XIVb (Cluster) Ascusbbr_2076 SEQID Paenibacillus Ascusbbr_2551 SEQ ID NO: 169 NO: 309 112.Butyricicoccus (Genus) Ascusbbr_2101 SEQ ID Olivibacter Ascusbbr_2553SEQ ID NO: 170 NO: 310 113. Pediococcus (Genus) Ascusbbr_2118 SEQ IDClostridium XII Ascusbbr_2554 SEQ ID NO: 171 NO: 311 114. Sphingomonas(Genus) Ascusbbr_2127 SEQ ID Sphingobacterium Ascusbbr_2555 SEQ ID NO:172 NO: 312 115. Clostridium XIVa (Cluster) Ascusbbr_2131 SEQ IDSphingobacterium Ascusbbr_2556 SEQ ID NO: 173 NO: 313 116. ClostridiumIV (Cluster) Ascusbbr_2132 SEQ ID Anaerosporobacter Ascusbbr_2557 SEQ IDNO: 174 NO: 314 117. Clostridium XIVb (Cluster) Ascusbbr_2136 SEQ IDClostridium XII Ascusbbr_2560 SEQ ID NO: 175 NO: 315 118. ClostridiumXIVb (Cluster) Ascusbbr_2137 SEQ ID Clostridium XII Ascusbbr_2561 SEQ IDNO: 176 NO: 316 119. Methylobacterium (Genus) Ascusbbr_2149 SEQ IDClostridium Ascusbbr_2562 SEQ ID NO: 177 NO: 317 120. Salana (Genus)Ascusbbr_2177 SEQ ID Pedobacter Ascusbbr_2563 SEQ ID NO: 178 NO: 318121. Petrobacter (Genus) Ascusbbr_2178 SEQ ID Bacillus Ascusbbr_2564 SEQID NO: 179 NO: 319 122. Bacillus (Genus) Ascusbbr_2180 SEQ IDPaenibacillus Ascusbbr_2565 SEQ ID NO: 180 NO: 320 123. Thermovibrio(Genus) Ascusbbr_2183 SEQ ID Prevotella Ascusbbr_2566 SEQ ID NO: 181 NO:321 124. Erysipelotrichaceae (Family) Ascusbbr_2184 SEQ IDLachnospiracea (Family) Ascusbbr_2567 SEQ ID NO: 182 NO: 322 125.Selenomonas (Genus) Ascusbbr_2192 SEQ ID Lachnospiracea (Family)Ascusbbr_2568 SEQ ID NO: 183 NO: 323 126. Glaciecola (Genus)Ascusbbr_2193 SEQ ID Escherichia/Shigella (Genus) Ascusbbr_2594 SEQ IDNO: 184 NO: 324 127. Lactobacillus (Genus) Ascusbbr_2195 SEQ IDLactobacillus (Genus) Ascusbbr_2603 SEQ ID NO: 185 NO: 325 128.Eubacterium (Genus) Ascusbbr_2200 SEQ ID Corynebacterium (Genus)Ascusbbr_2605 SEQ ID NO: 186 NO: 326 129. Thermomicrobium (Genus)Ascusbbr_2201 SEQ ID Lactobacillus (Genus) Ascusbbr_2615 SEQ ID NO: 187NO: 327 130. Acidobacteria (Genus) Ascusbbr_2204 SEQ ID Lactobacillus(Genus) Ascusbbr_2625 SEQ ID NO: 188 NO: 328 131. Chlorobaculum (Genus)Ascusbbr_2205 SEQ ID Escherichia/Shigella (Genus) Ascusbbr_2640 SEQ IDNO: 189 NO: 329 132. Rothia (Genus) Ascusbbr_2208 SEQ ID Lactobacillus(Genus) Ascusbbr_2644 SEQ ID NO: 190 NO: 330 133. Selenomonas (Genus)Ascusbbr_2210 SEQ ID Lactobacillus (Genus) Ascusbbr_2665 SEQ ID NO: 191NO: 331 134. Clostridium XIVa (Cluster) Ascusbbr_2215 SEQ IDLactobacillus (Genus) Ascusbbr_2684 SEQ ID NO: 192 NO: 332 135.Virgibacillus (Genus) Ascusbbr_2216 SEQ ID Lactobacillus (Genus)Ascusbbr_2694 SEQ ID NO: 193 NO: 333 136. Sphingomonas (Genus)Ascusbbr_2218 SEQ ID Lactobacillus (Genus) Ascusbbr_2699 SEQ ID NO: 194NO: 334 137. Citricoccus (Genus) Ascusbbr_2219 SEQ ID Lactobacillus(Genus) Ascusbbr_2709 SEQ ID NO: 195 NO: 335 138. Catenibacterium(Genus) Ascusbbr_2220 SEQ ID Lactobacillus (Genus) Ascusbbr_2710 SEQ IDNO: 196 NO: 336 139. Amycolatopsis (Genus) Ascusbbr_2224 SEQ IDEnterococcus (Genus) Ascusbbr_2714 SEQ ID NO: 197 NO: 337 140.Sphingobium (Genus) Ascusbbr_2225 SEQ ID Clostridium sensu strictoAscubbr_2676 SEQ ID NO: 198 (Genus) NO: 386 141. Clostridium_XIVa(Cluster) Ascusbbr_105932 SEQ ID NO: 387

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a general workflow of one embodiment of the method fordetermining the absolute abundance of one or more active microorganismstrains.

FIG. 2 shows a general workflow of one embodiment of a method fordetermining the co-occurrence of one or more, or two or more, activemicroorganism strains in a sample with one or more metadata(environmental) parameters, followed by leveraging cluster analysis andcommunity detection methods on the network of determined relationships.

FIG. 3 is a cartoon depiction of an exemplary chicken's anatomy.

FIG. 4 is an image of a dissected gastrointestinal track of a chickenfrom the beak to the cloaca.

FIG. 5 depicts the complex microbial interactions occurring in thegastrointestinal tract. A well-balanced commensal microbial load isinvolved in maintaining multiple homeostatic systems.

FIG. 6 depicts the likely results of a lectin binding assay withisolated microbial strains and a mannose-binding lectin (MBL).

FIG. 7 depicts the likely results of a gelatin/collagen binding assaywith isolated microbial strains. CP=Clostridium perfringens, Strain1=avirulent cnaA positive, Strain 2=avirulent cnaA negative. Allreported results were corrected by their respective controls

FIG. 8 depicts a histogram the rate of microbial convergence across theexperimental groups.

FIG. 9 depicts a histogram of the final percentage mortality for theexperiment.

DETAILED DESCRIPTION Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

The term “a” or “an” may refer to one or more of that entity, i.e. canrefer to plural referents. As such, the terms “a” or “an”, “one or more”and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

Reference throughout this specification to “one embodiment”, “anembodiment”, “one aspect”, or “an aspect” means that a particularfeature, structure or characteristic described in connection with theembodiment is included in at least one embodiment of the presentdisclosure. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics can be combined inany suitable manner in one or more embodiments.

As used herein, in particular embodiments, the terms “about” or“approximately” when preceding a numerical value indicates the valueplus or minus a range of 10%.

As used herein the terms “microorganism” or “microbe” should be takenbroadly. These terms are used interchangeably and include, but are notlimited to, the two prokaryotic domains, Bacteria and Archaea,eukaryotic fungi and protists, as well as viruses. In some embodiments,the disclosure refers to the “microbes” of Table 1 and/or Table 3, orthe “microbes” incorporated by reference. This characterization canrefer to not only the predicted taxonomic microbial identifiers of thetable, but also the identified strains of the microbes listed in thetable.

The term “bioensemble,” “microbial ensemble,” or “synthetic ensemble”refers to a composition comprising one or more active microbesidentified by methods, systems, and/or apparatuses of the presentdisclosure and that do not naturally exist in a naturally occurringenvironment and/or at ratios or amounts that do not exist in nature. Abioensemble is a subset of a microbial community of individual microbialspecies, or strains of a species, which can be described as carrying outa common function, or can be described as participating in, or leadingto, or correlating with, a recognizable parameter, such as a phenotypictrait of interest (e.g. increased feed efficiency in poultry). Thebioensemble may comprise two or more species, or strains of a species,of microbes. In some instances, the microbes coexist within thecommunity symbiotically.

In certain aspects of the disclosure, bioensembles are or are based onone or more isolated microbes that exist as isolated and biologicallypure cultures. It will be appreciated by one of ordinary skill in theart that an isolated and biologically pure culture of a particularmicrobe, denotes that said culture is substantially free (withinscientific reason) of other living organisms and contains only theindividual microbe in question. The culture can contain varyingconcentrations of said microbe. The present disclosure notes thatisolated and biologically pure microbes often “necessarily differ fromless pure or impure materials.” See, e.g. In re Bergstrom, 427 F.2d1394, (CCPA 1970) (discussing purified prostaglandins), see also, In reBergy, 596 F.2d 952 (CCPA 1979) (discussing purified microbes), seealso, Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911)(J. Hand discussing purified adrenaline, aff'd in part, rev'd in part,196 F. 496 (2d Cir. 1912), each of which are incorporated herein byreference. Furthermore, in some aspects, implementation of thedisclosure can require certain quantitative measures of theconcentration or purity limitations that must be achieved for anisolated and biologically pure microbial culture to be used in thedisclosed microbial ensembles. The presence of these purifty values, incertain embodiments, is a further attribute that distinguishes themicrobes identified by the presently disclosed method from thosemicrobes existing in a natural state. See, e.g., Merck & Co. v. OlinMathieson Chemical Corp., 253 F.2d 156 (4t Cir. 1958) (discussing puritylimitations for vitamin B12 produced by microbes), incorporated hereinby reference.

Bioensembles can be applied or administered to a target, such as atarget environment, population, individual, animal, and/or the like.

The term “microbial community” means a group of microbes comprising twoor more species or strains. Unlike bioensembles, a microbial communitydoes not have to be carrying out a common function, or does not have tobe participating in, or leading to, or correlating with, a recognizableparameter, such as a phenotypic trait of interest (e.g. increased feedefficiency in poultry).

As used herein, “isolate,” “isolated,” “isolated microbe,” and liketerms, are intended to mean that the one or more microorganisms has beenseparated from at least one of the materials with which it is associatedin a particular environment (for example soil, water, animal tissue).

Microbes of the present disclosure may include spores and/or vegetativecells. In some embodiments, microbes of the present disclosure includemicrobes in a viable but non-culturable (VBNC) state, or a quiescentstate. See Liao and Zhao (US Publication US2015267163A1). In someembodiments, microbes of the present disclosure include microbes in abiofilm. See Merritt et al. (U.S. Pat. No. 7,427,408).

Thus, an “isolated microbe” does not exist in its naturally occurringenvironment; rather, it is through the various techniques describedherein that the microbe has been removed from its natural setting andplaced into a non-naturally occurring state of existence. Thus, theisolated strain or isolated microbe may exist as, for example, abiologically pure culture, or as spores (or other forms of the strain)in association with an acceptable carrier.

As used herein, “spore” or “spores” refer to structures produced bybacteria and fungi that are adapted for survival and dispersal. Sporesare generally characterized as dormant structures; however, spores arecapable of differentiation through the process of germination.Germination is the differentiation of spores into vegetative cells thatare capable of metabolic activity, growth, and reproduction. Thegermination of a single spore results in a single fungal or bacterialvegetative cell. Fungal spores are units of asexual reproduction, and insome cases are necessary structures in fungal life cycles. Bacterialspores are structures for surviving conditions that may ordinarily benonconductive to the survival or growth of vegetative cells.

As used herein, “microbial composition” refers to a compositioncomprising one or more microbes of the present disclosure, wherein amicrobial composition, in some embodiments, is administered to animalsof the present disclosure.

As used herein, “carrier”, “acceptable carrier”, or “pharmaceuticalcarrier” refers to a diluent, adjuvant, excipient, or vehicle with whichthe compound is administered. Such carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable, orsynthetic origin; such as peanut oil, soybean oil, mineral oil, sesameoil, and the like. Water or aqueous solution saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, in some embodiments as injectable solutions. In someembodiments, gelling agents are employed as carriers. Alternatively, thecarrier can be a solid dosage form carrier, including but not limited toone or more of a binder (for compressed pills), a glidant, anencapsulating agent, a flavorant, and a colorant. The choice of carriercan be selected with regard to the intended route of administration andstandard pharmaceutical practice. See Hardee and Baggo (1998.Development and Formulation of Veterinary Dosage Forms. 2^(nd) Ed. CRCPress. 504 pg.); E. W. Martin (1970. Remington's PharmaceuticalSciences. 17^(th) Ed. Mack Pub. Co.); and Blaser et al. (US PublicationUS20110280840A1).

In some aspects, carriers may be granular in structure, such as sand orsand particles. In further aspects, the carriers may be dry, as opposedto a moist or wet carrier. In some aspects, carriers can be nutrititvesubstances and/or prebiotic substances selected fromfructo-oligosaccharides, inulins, isomalto-oligosaccharides, lactitol,lactosucruse, lactulose, pyrodextrines, soy oligosaccharides,transgalacto-oligosaccharides, xylo-oligosaccharides, trace minerals,and vitamins. In some aspects, carriers can be in solid or liquid form.In some aspects, carriers can be zeolites, calcium carbonate, magnesiumcarbonate, silicon dioxide, ground corn, trehalose, chitosan, shellac,albumin, starch, skim-milk powder, sweet-whey powder, maltodextrin,lactose, and inulin. In some aspects, a carrier is water orphysiological saline.

In certain aspects of the disclosure, the isolated microbes exist asisolated and biologically pure cultures. It will be appreciated by oneof skill in the art, that an isolated and biologically pure culture of aparticular microbe, denotes that said culture is substantially free(within scientific reason) of other living organisms and contains onlythe individual microbe in question. The culture can contain varyingconcentrations of said microbe. The present disclosure notes thatisolated and biologically pure microbes often “necessarily differ fromless pure or impure materials.” See, e.g. In re Bergstrom, 427 F.2d1394, (CCPA 1970)(discussing purified prostaglandins), see also, In reBergy, 596 F.2d 952 (CCPA 1979)(discussing purified microbes), see also,Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911)(Learned Hand discussing purified adrenaline), aff'd in part, rev'd inpart, 196 F. 496 (2d Cir. 1912), each of which are incorporated hereinby reference. Furthermore, in some aspects, the disclosure provides forcertain quantitative measures of the concentration, or puritylimitations, that must be found within an isolated and biologically puremicrobial culture. The presence of these purity values, in certainembodiments, is a further attribute that distinguishes the presentlydisclosed microbes from those microbes existing in a natural state. See,e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4thCir. 1958) (discussing purity limitations for vitamin B12 produced bymicrobes), incorporated herein by reference.

As used herein, “individual isolates” should be taken to mean acomposition, or culture, comprising a predominance of a single genera,species, or strain, of microorganism, following separation from one ormore other microorganisms. The phrase should not be taken to indicatethe extent to which the microorganism has been isolated or purified.However, “individual isolates” can comprise substantially only onegenus, species, or strain, of microorganism.

As used herein, “microbiome” refers to the collection of microorganismsthat inhabit the reproductive tract, integument system, digestive tractor gastrointestinal tract of an animal and the microorganisms' physicalenvironment (i.e., the microbiome has a biotic and physical component).The microbiome is fluid and may be modulated by numerous naturallyoccurring and artificial conditions (e.g., change in diet, disease,antimicrobial agents, influx of additional microorganisms, etc.). Themodulation of the gastrointestinal microbiome can be achieved viaadministration of the compositions of the disclosure can take the formof: (a) increasing or decreasing a particular Family, Genus, Species, orfunctional grouping of a microbe (i.e., alteration of the bioticcomponent of the gastrointestinal microbiome) and/or (b) increasing ordecreasing gastrointestinal pH, increasing or decreasing volatile fattyacids in the gastrointestinal tract, increasing or decreasing any otherphysical parameter important for gastrointestinal health (i.e.,alteration of the abiotic component of the gut microbiome).

As used herein, “probiotic” refers to a substantially pure microbe(i.e., a single isolate) or a mixture of desired microbes, and may alsoinclude any additional components that can be administered to poultryfor restoring microbiota. Probiotics or microbial inoculant compositionsof the invention may be administered with an agent to allow the microbesto survive the environment of the gastrointestinal tract, i.e., toresist low pH and to grow in the gastrointestinal environment. In someembodiments, the present compositions (e.g., microbial compositions) areprobiotics in some aspects.

As used herein, “prebiotic” refers to an agent that increases the numberand/or activity of one or more desired microbes. Non-limiting examplesof prebiotics that may be useful in the methods of the presentdisclosure include fructooligosaccharides (e.g., oligofructose, inulin,inulin-type fructans), galactooligosaccharides, amino acids, alcohols,isomalto-oligosaccharides, lactitol, lactosucruse, lactulose,pyrodextrines, soy oligosaccharides, transgalacto-oligosaccharides,xylo-oligosaccharides, vitamins, and mixtures thereof. SeeRamirez-Farias et al. (2008. Br. J. Nutr. 4:1-10) and Pool-Zobel andSauer (2007. J. Nutr. 137:2580-2584 and supplemental).

The term “growth medium” as used herein, is any medium which is suitableto support growth of a microbe. By way of example, the media may benatural or artificial including gastrin supplemental agar, LB media,blood serum, and tissue culture gels. It should be appreciated that themedia may be used alone or in combination with one or more other media.It may also be used with or without the addition of exogenous nutrients.

The medium may be amended or enriched with additional compounds orcomponents, for example, a component which may assist in the interactionand/or selection of specific groups of microorganisms. For example,antibiotics (such as penicillin) or sterilants (for example, quaternaryammonium salts and oxidizing agents) could be present and/or thephysical conditions (such as salinity, nutrients (for example organicand inorganic minerals (such as phosphorus, nitrogenous salts, ammonia,potassium and micronutrients such as cobalt and magnesium), pH, and/ortemperature) could be amended.

As used herein, the term “fowl” and “poultry” are used interchangeablyto include both domesticated and non-domesticated birds belonging to theorders of Galliformes and Anseriformes. Fowl include chickens(broilers/fryers/roasters/capons/roosters/stewing hens), turkeys,grouse, New World quail, Old World quail, partridges, ptarmigans,junglefowl, peafowl, ducks, geese, swans, emus, and ostriches.

As used herein, “improved” should be taken broadly to encompassimprovement of a characteristic of interest, as compared to a controlgroup, or as compared to a known average quantity associated with thecharacteristic in question. For example, “improved” feed efficiencyassociated with application of a beneficial microbe, or bioensembles, ofthe disclosure can be demonstrated by comparing the feed efficiency ofpoultry treated by the microbes taught herein to the feed efficiency ofpoultry not treated. In the present disclosure, “improved” does notnecessarily demand that the data be statistically significant (i.e.p<0.05); rather, any quantifiable difference demonstrating that onevalue (e.g. the average treatment value) is different from another (e.g.the average control value) can rise to the level of “improved.”

As used herein, “inhibiting and suppressing” and like terms should notbe construed to require complete inhibition or suppression, althoughthis may be desired in some embodiments.

The term “marker” or “unique marker” as used herein is an indicator ofunique microorganism type, microorganism strain or activity of amicroorganism strain. A marker can be measured in biological samples andincludes without limitation, a nucleic acid-based marker such as aribosomal RNA gene, a peptide- or protein-based marker, and/or ametabolite or other small molecule marker.

The term “metabolite” as used herein is an intermediate or product ofmetabolism. A metabolite in one embodiment is a small molecule.Metabolites have various functions, including in fuel, structural,signaling, stimulatory and inhibitory effects on enzymes, as a cofactorto an enzyme, in defense, and in interactions with other organisms (suchas pigments, odorants and pheromones). A primary metabolite is directlyinvolved in normal growth, development and reproduction. A secondarymetabolite is not directly involved in these processes but usually hasan important ecological function. Examples of metabolites include butare not limited to antibiotics and pigments such as resins and terpenes,etc. Some antibiotics use primary metabolites as precursors, such asactinomycin which is created from the primary metabolite, tryptophan.Metabolites, as used herein, include small, hydrophilic carbohydrates;large, hydrophobic lipids and complex natural compounds.

As used herein, the term “genotype” refers to the genetic makeup of anindividual cell, cell culture, tissue, organism, or group of organisms.

As used herein, the term “allele(s)” means any of one or morealternative forms of a gene, all of which alleles relate to at least onetrait or characteristic. In a diploid cell, the two alleles of a givengene occupy corresponding loci on a pair of homologous chromosomes.Since the present disclosure, in embodiments, relates to QTLs, i.e.genomic regions that may comprise one or more genes or regulatorysequences, it is in some instances more accurate to refer to “haplotype”(i.e. an allele of a chromosomal segment) instead of “allele”, however,in those instances, the term “allele” should be understood to comprisethe term “haplotype”. Alleles are considered identical when they expressa similar phenotype. Differences in sequence are possible but notimportant as long as they do not influence phenotype.

As used herein, the term “locus” (loci plural) means a specific place orplaces or a site on a chromosome where for example a gene or geneticmarker is found.

As used herein, the term “genetically linked” refers to two or moretraits that are co-inherited at a high rate during breeding such thatthey are difficult to separate through crossing.

A “recombination” or “recombination event” as used herein refers to achromosomal crossing over or independent assortment. The term“recombinant” refers to an organism having a new genetic makeup arisingas a result of a recombination event.

As used herein, the term “molecular marker” or “genetic marker” refersto an indicator that is used in methods for visualizing differences incharacteristics of nucleic acid sequences. Examples of such indicatorsare restriction fragment length polymorphism (RFLP) markers, amplifiedfragment length polymorphism (AFLP) markers, single nucleotidepolymorphisms (SNPs), insertion mutations, microsatellite markers(SSRs), sequence-characterized amplified regions (SCARs), cleavedamplified polymorphic sequence (CAPS) markers or isozyme markers orcombinations of the markers described herein which defines a specificgenetic and chromosomal location. Markers further include polynucleotidesequences encoding 16S or 18S rRNA, and internal transcribed spacer(ITS) sequences, which are sequences found between small-subunit andlarge-subunit rRNA genes that have proven to be especially useful inelucidating relationships or distinctions among when compared againstone another. Mapping of molecular markers in the vicinity of an alleleis a procedure which can be performed by the average person skilled inmolecular-biological techniques.

The primary structure of major rRNA subunit 16S comprise a particularcombination of conserved, variable, and hypervariable regions thatevolve at different rates and enable the resolution of both very ancientlineages such as domains, and more modern lineages such as genera. Thesecondary structure of the 16S subunit include approximately 50 heliceswhich result in base pairing of about 67% of the residues. These highlyconserved secondary structural features are of great functionalimportance and can be used to ensure positional homology in multiplesequence alignments and phylogenetic analysis. Over the previous fewdecades, the 16S rRNA gene has become the most sequenced taxonomicmarker and is the cornerstone for the current systematic classificationof bacteria and archaea (Yarza et al. 2014. Nature Rev. Micro.12:635-45).

A sequence identity of 94.5% or lower for two 16S rRNA genes is strongevidence for distinct genera, 86.5% or lower is strong evidence fordistinct families, 82% or lower is strong evidence for distinct orders,78.5% is strong evidence for distinct classes, and 75% or lower isstrong evidence for distinct phyla. The comparative analysis of 16S rRNAgene sequences enables the establishment of taxonomic thresholds thatare useful not only for the classification of cultured microorganismsbut also for the classification of the many environmental sequences.Yarza et al. 2014. Nature Rev. Micro. 12:635-45).

As used herein, the term “trait” refers to a characteristic orphenotype. For example, in the context of some embodiments of thepresent disclosure; quantity of eggs produced, efficiency of feedutilization, amount of feces produced, susceptibility to gut pathogens,and a decrease in mortality rates, among others. Desirable traits mayalso include other characteristics, including but not limited to: anincrease in weight; an increase in egg production; an increase ofmusculature; an increase of vitamins in eggs; an increase of fatty acidconcentration in the gastrointestinal tract; and increase in egg volume;an improved efficiency in feed utilization and digestibility; anincrease in polysaccharide and lignin degradation; an increase in fat,starch, and protein digestion; an increase in vitamin availability; anincrease in mineral availability; an increase in amino acidavailability; improved gastrointestinal development; increasing villilength and surface area; pH balance in the gastrointestinal tract; pHincrease in the gastrointestinal tract, pH decrease in thegastrointestinal tract, a reduction in methane and/or nitrous oxideemissions; a reduction in manure production; an improved efficiency ofnitrogen utilization; an improved efficiency of phosphorous utilization;an increased resistance to colonization of pathogenic microbes thatcolonize chickens; an improvement in meat properties, reduced mortality,increased production of antimicrobials, increased clearance ofpathogenic microbes, increased resistance to colonization of pathogenicmicrobes that infect chickens, increased resistance to colonization ofpathogenic microbes that infect humans improved gut health, etc.;wherein said increase, decrease, or reduction is determined by comparingagainst an animal not having been administered a composition of thepresent disclosure.

A trait may be inherited in a dominant or recessive manner, or in apartial or incomplete-dominant manner. A trait may be monogenic (i.e.determined by a single locus) or polygenic (i.e. determined by more thanone locus) or may also result from the interaction of one or more geneswith the environment.

In the context of this disclosure, traits may also result from theinteraction of one or more avian genes and one or more microorganismgenes.

As used herein, the term “homozygous” means a genetic condition existingwhen two identical alleles reside at a specific locus, but arepositioned individually on corresponding pairs of homologous chromosomesin the cell of a diploid organism. Conversely, as used herein, the term“heterozygous” means a genetic condition existing when two differentalleles reside at a specific locus, but are positioned individually oncorresponding pairs of homologous chromosomes in the cell of a diploidorganism.

As used herein, the term “phenotype” refers to the observablecharacteristics of an individual cell, cell culture, organism (e.g.,bird), or group of organisms which results from the interaction betweenthat individual's genetic makeup (i.e., genotype) and the environment.

As used herein, the term “chimeric” or “recombinant” when describing anucleic acid sequence or a protein sequence refers to a nucleic acid, ora protein sequence, that links at least two heterologouspolynucleotides, or two heterologous polypeptides, into a singlemacromolecule, or that re-arranges one or more elements of at least onenatural nucleic acid or protein sequence. For example, the term“recombinant” can refer to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques.

As used herein, a “synthetic nucleotide sequence” or “syntheticpolynucleotide sequence” is a nucleotide sequence that is not known tooccur in nature or that is not naturally occurring. Generally, such asynthetic nucleotide sequence will comprise at least one nucleotidedifference when compared to any other naturally occurring nucleotidesequence.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, the term “gene” refers to any segment of DNA associatedwith a biological function. Thus, genes include, but are not limited to,coding sequences and/or the regulatory sequences required for theirexpression. Genes can also include non-expressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

As used herein, the term “homologous” or “homologue” or “ortholog” isknown in the art and refers to related sequences that share a commonancestor or family member and are determined based on the degree ofsequence identity. The terms “homology,” “homologous,” “substantiallysimilar” and “corresponding substantially” are used interchangeablyherein. They refer to nucleic acid fragments wherein changes in one ormore nucleotide bases do not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant disclosure such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the disclosure encompasses more than the specificexemplary sequences. These terms describe the relationship between agene found in one species, subspecies, variety, cultivar or strain andthe corresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this disclosure homologoussequences are compared. “Homologous sequences” or “homologues” or“orthologs” are thought, believed, or known to be functionally related.A functional relationship may be indicated in any one of a number ofways, including, but not limited to: (a) degree of sequence identityand/or (b) the same or similar biological function. Preferably, both (a)and (b) are indicated. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus(Scientific and Educational Software, Pennsylvania) and AlignX (VectorNTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher(Gene Codes, Ann Arbor, Michigan), using default parameters.

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule. Afragment of a polynucleotide of the disclosure may encode a biologicallyactive portion of a genetic regulatory element. A biologically activeportion of a genetic regulatory element can be prepared by isolating aportion of one of the polynucleotides of the disclosure that comprisesthe genetic regulatory element and assessing activity as describedherein. Similarly, a portion of a polypeptide may be 4 amino acids, 5amino acids, 6 amino acids, 7 amino acids, and so on, going up to thefull length polypeptide. The length of the portion to be used willdepend on the particular application. A portion of a nucleic acid usefulas a hybridization probe may be as short as 12 nucleotides; in someembodiments, it is 20 nucleotides. A portion of a polypeptide useful asan epitope may be as short as 4 amino acids. A portion of a polypeptidethat performs the function of the full-length polypeptide wouldgenerally be longer than 4 amino acids.

Variant polynucleotides also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. Strategiesfor such DNA shuffling are known in the art. See, for example, Stemmer(1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameriet al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.Biol. 272:336-347; Zhang et al. (1997) PNAS 94:4504-4509; Crameri et al.(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.For PCR amplifications of the polynucleotides disclosed herein,oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from cDNA or genomic DNA extractedfrom any organism of interest. Methods for designing PCR primers and PCRcloning are generally known in the art and are disclosed in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold SpringHarbor Laboratory Press, Plainview, New York). See also Innis et al.,eds. (1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T vs. G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

The terms “stringency” or “stringent hybridization conditions” refer tohybridization conditions that affect the stability of hybrids, e.g.,temperature, salt concentration, pH, formamide concentration and thelike. These conditions are empirically optimized to maximize specificbinding and minimize non-specific binding of primer or probe to itstarget nucleic acid sequence. The terms as used include reference toconditions under which a probe or primer will hybridize to its targetsequence, to a detectably greater degree than other sequences (e.g. atleast 2-fold over background). Stringent conditions are sequencedependent and will be different in different circumstances. Longersequences hybridize specifically at higher temperatures. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of a complementary target sequence hybridizes to aperfectly matched probe or primer. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M Na+ion, typically about 0.01 to 1.0 M Na+ ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes or primers (e.g. 10 to 50 nucleotides) and at least about60° C. for long probes or primers (e.g. greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. Exemplary low stringentconditions or “conditions of reduced stringency” include hybridizationwith a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 370 C and awash in 2×SSC at 400 C. Exemplary high stringency conditions includehybridization in 50% formamide, 1M NaCl, 1% SDS at 370 C, and a wash in0.1×SSC at 600 C. Hybridization procedures are well known in the art andare described by e.g. Ausubel et al., 1998 and Sambrook et al., 2001. Insome embodiments, stringent conditions are hybridization in 0.25 MNa2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5-20% sodium dodecylsulfate at 45° C., such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by awash in 5×SSC, containing 0.1% (w/v) sodium dodecyl sulfate, at 55° C.to 65° C.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Thepromoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic DNA segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of some variation may have identicalpromoter activity.

As used herein, a “constitutive promoter” is a promoter which is activeunder most conditions and/or during most development stages. There areseveral advantages to using constitutive promoters in expression vectorsused in biotechnology, such as: high level of production of proteinsused to select transgenic cells or organisms; high level of expressionof reporter proteins or scorable markers, allowing easy detection andquantification; high level of production of a transcription factor thatis part of a regulatory transcription system; production of compoundsthat requires ubiquitous activity in the organism; and production ofcompounds that are required during all stages of development.Non-limiting exemplary constitutive promoters include, CaMV 35Spromoter, opine promoters, ubiquitin promoter, alcohol dehydrogenasepromoter, etc.

As used herein, a “non-constitutive promoter” is a promoter which isactive under certain conditions, in certain types of cells, and/orduring certain development stages. For example, tissue specific, tissuepreferred, cell type specific, cell type preferred, inducible promoters,and promoters under development control are non-constitutive promoters.Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues.

As used herein, “inducible” or “repressible” promoter is a promoterwhich is under chemical or environmental factors control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions, certain chemicals, the presenceof light, acidic or basic conditions, etc.

As used herein, a “tissue specific” promoter is a promoter thatinitiates transcription only in certain tissues. Unlike constitutiveexpression of genes, tissue-specific expression is the result of severalinteracting levels of gene regulation. As such, in the art sometimes itis preferable to use promoters from homologous or closely relatedspecies to achieve efficient and reliable expression of transgenes inparticular tissues. This is one of the main reasons for the large amountof tissue-specific promoters isolated from particular tissues found inboth scientific and patent literature.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is regulated by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of regulatingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of thedisclosure can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

As used herein, the phrases “recombinant construct”, “expressionconstruct”, “chimeric construct”, “construct”, and “recombinant DNAconstruct” are used interchangeably herein. A recombinant constructcomprises an artificial combination of nucleic acid fragments, e.g.,regulatory and coding sequences that are not found together in nature.For example, a chimeric construct may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such constructmay be used by itself or may be used in conjunction with a vector. If avector is used then the choice of vector is dependent upon the methodthat will be used to transform host cells as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the disclosure. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others. Vectors can be plasmids, viruses,bacteriophages, pro-viruses, phagemids, transposons, artificialchromosomes, and the like, that replicate autonomously or can integrateinto a chromosome of a host cell. A vector can also be a naked RNApolynucleotide, a naked DNA polynucleotide, a polynucleotide composed ofboth DNA and RNA within the same strand, a poly-lysine-conjugated DNA orRNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or thelike, that is not autonomously replicating. As used herein, the term“expression” refers to the production of a functional end-product e.g.,an mRNA or a protein (precursor or mature).

In some embodiments, the cell or organism has at least one heterologoustrait. As used herein, the term “heterologous trait” refers to aphenotype imparted to a transformed host cell or transgenic organism byan exogenous DNA segment, heterologous polynucleotide or heterologousnucleic acid. Various changes in phenotype are of interest to thepresent disclosure, including but not limited to increasing a fowl'syield of an economically important trait (e.g., eggs, egg volume,poultry weight, etc.) and the like. These results can be achieved byproviding expression of heterologous products or increased expression ofendogenous products in organisms using the methods and compositions ofthe present disclosure. In some embodiments, the isolated microbialstrains of the present disclosure further encompass mutants thereof. Insome embodiments, the present disclosure further contemplates microbialstrains having all of the identifying characteristics of the presentlydisclosed microbial strains.

As used herein, the term “MIC” means maximal information coefficient.MIC is a type of nonparamentric analysis that identifies a score (MICscore) between active microbial strains of the present disclosure and atleast one measured metadata (e.g., increase in weight). Further, U.S.application Ser. No. 15/217,575, filed on Jul. 22, 2016 (issued as U.S.Pat. No. 9,540,676 on Jan. 10, 2017) is hereby incorporated by referencein its entirety.

The maximal information coefficient (MIC) is then calculated betweenstrains and metadata and between strains as seen in FIG. 2, 2009 .Results are pooled to create a list of all relationships and theircorresponding MIC scores. If the relationship scores below a giventhreshold, the relationship is deemed/identified as irrelevant. If therelationship is above a given threshold, the relationshipdeemed/identified as relevant, and is further subject to networkanalysis. The following code fragment shows an exemplary methodology forsuch analysis, according to one embodiment:

Read total list of relationships file as links threshold = 0.8 for i inrange(len(link)):  if links >= threshold   mulitiplier[i] = 1  else  mulitiplier[i] = 0 end if link_temp = multiplier*links final_links =links_temp[links_temp != 0] savetxt(output_file,final_links)output_file.close( )

In some embodiments, the compositions of the present disclosure compriseone or more bacteria and/or one or more fungus that have a MIC score ofat least about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55,0.6, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95.

With regard to MIC scores a cut-off based on this score may be used todefine useful and non-useful microorganisms with respect to theimprovement of specific traits. The point at which data points on acurve transition from a log scale to a linear scale (with regard to theslope) is the inflection point. Organisms with MIC scores that fallbelow the inflection point are generally non-useful, while the organismswith MIC scores that are found above the inflection point are generallyuseful, as it pertains to the specific characteristic being evaluatedfor the MIC score.

Based on the output of the network analysis, active strains are selectedfor preparing products (e.g., ensembles, aggregates, and/or othersynthetic groupings) containing the selected strains. The output of thenetwork analysis can also be used to inform the selection of strains forfurther product composition testing, as seen in FIG. 2, 2010 .

The use of thresholds is discussed above for analyses anddeterminations. Thresholds can be, depending on the implementation andapplication: (1) empirically determined (e.g., based on distributionlevels, setting a cutoff at a number that removes a specified orsignificant portion of low level reads); (2) any non-zero value; (3)percentage/percentile based; (4) only strains whose normalized secondmarker (i.e., activity) reads is greater than normalized first marker(cell count) reads; (5) log 2 fold change between activity and quantityor cell count; (6) normalized second marker (activity) reads is greaterthan mean second marker (activity) reads for entire sample (and/orsample set); and/or any magnitude threshold described above in additionto a statistical threshold (i.e., significance testing). The followingexample provides thresholding detail for distributions of RNA-basedsecond marker measurements with respect to DNA-based first markermeasurements, according to one embodiment.

As used herein “shelf-stable” refers to a functional attribute and newutility acquired by the microbes formulated according to the disclosure,which enable said microbes to exist in a useful/active state outside oftheir natural environment in the gastrointestinal tract (i.e. a markedlydifferent characteristic). Thus, shelf-stable is a functional attributecreated by the formulations/compositions of the disclosure and denotingthat the microbe formulated into a shelf-stable composition can existoutside the gastrointestinal tract and under ambient conditions for aperiod of time that can be determined depending upon the particularformulation utilized, but in general means that the microbes can beformulated to exist in a composition that is stable under ambientconditions for at least a few days and generally at least one week.Accordingly, a “shelf-stable poultry supplement” is a compositioncomprising one or more microbes of the disclosure, said microbesformulated in a composition, such that the composition is stable underambient conditions for at least one week, meaning that the microbescomprised in the composition (e.g. whole cell, spore, or lysed cell) areable to impart one or more beneficial phenotypic properties to poultrywhen administered (e.g. increased weight gain, increased eggshelldensity, improved gastrointestinal health, and/or modulation of thegastrointestinal microbiome)

In aspects, the aforementioned microbial species—that is, a purifiedmicrobial population that comprises a bacteria with a 16S nucleic acidsequence, and/or a fungi with an ITS nucleic acid sequence, which is atleast about 97% identical to a nucleic acid sequence selected from thegroup consisting of: SEQ ID NOs: 1-387—are members of a Markush group,as the present disclosure illustrates that the members belong to a classof microbes characterized by various physical and functional attributes,which can include any of the following: a) the ability to convert acarbon source into a volatile fatty acid such as acetate, butyrate,propionate, or combinations thereof, b) the ability to degrade a solubleor insoluble carbon source; c) the ability to impart an increase inweight gain to poultry administered the microbe(s); d) the ability tomodulate the microbiome of the gastrointestinal tract of poultryadministered the microbe; e) the ability to be formulated into ashelf-stable composition; f) the ability to exhibit a decrease in feedconversion ratio in poultry having been administered the microbe(s); g)the ability to impart a decrease in pathogen-associated lesion formationin the gastrointestinal tract; h) the ability to impart a decrease inpathogenic microbes in the gastrointestinal tract; and/or i) possessinga MIC score of at least about 0.2 if a bacteria and possessing a MICscore of at least about 0.2 if a fungi. Thus, the members of the Markushgroup possess at least one property in common, which can be responsiblefor their function in the claimed relationship.

In some embodiments, the isolated microbial strains of the presentdisclosure further encompass mutants thereof. In some embodiments, thepresent disclosure further contemplates microbial strains having all ofthe identifying characteristics of the presently disclosed microbialstrains.

Isolated Microbes

In some aspects, the present disclosure provides isolated microbes,including novel strains of microbes, presented in Table 1 and Table 3.

In other aspects, the present disclosure provides isolated wholemicrobial cultures of the microbes identified in Table 1 and Table 3.These cultures may comprise microbes at various concentrations.

In some aspects, the disclosure provides for utilizing one or moremicrobes selected from Table 1 and Table 3 to increase a phenotypictrait of interest in poultry.

In some embodiments, the disclosure provides isolated microbial speciesbelonging to taxonomic families of Lactobacillaceae, Lachnospiraceae,Ruminococcaceae, Peptostreptococcaceae, Streptosporangiaceae,Leuconostocaceae, Microbacteriaceae, Micromonosporaceae, Clostridiaceae,Pseudomonadaceae, Streptococcaceae, Bacillaceae, Bacteroidaceae,Nectriaceae, Corynebacteriaceae, Rhodobacteraceae, and Hypocreaceae.

In further embodiments, isolated microbial species may be selected fromgenera of family Lactobacillaceae, including Lactobacillus, Pediococcus,Paralactobacillus, and Sharpea.

In further embodiments, isolated microbial species may be selected fromgenera of family Lachnospiraceae, including Butyrivibrio, Roseburia,Lachnospira, Acetitomaculum, Coprococcus, Johnsonella, Catonella,Pseudobutyrivibrio, Syntrophococcus, Sporobacterium, Parasporobacterium,Lachnobacterium, Shuttleworthia, Dorea, Anaerostipes, Hespellia,Marvinbryantia, Oribacterium, Moryella, Blautia, Robinsoniella,Cellulosilyticum, Lachnoanaerobaculum, Stomatobaculum, Fusicatenibacter,Acetatifactor, and Eisenbergiella.

In further embodiments, isolated microbial species may be selected fromgenera of family Ruminococcaceae, including Ruminococcus, Acetivibrio,Sporobacter, Anaerofilium, Papillibacter, Oscillospira, Gemmiger,Faecalibacterium, Fastidiosipila, Anaerotruncus, Ethanolingenens,Acetanaerobacterium, Subdoligranulum, Hydrogenoanaerobacterium, andCandidadus Soleaferrea.

In further embodiments, isolated microbial species may be selected fromgenera of family Peptostreptococcaceae, including Anaerosphaera,Filifactor, Peptostreptococcus, Sporacetigenium, and Tepidibacter.

In further embodiments, isolated microbial species may be selected fromgenera of family Streptosporangiaceae, including Acrocarpospora,Herbidospora, Microbispora, Microtetraspora, Nonomuraea, Planobispora,Planomonospora, Planotetraspora, Sphaerisporangium, Streptosporangium,Thermoactinospora, Thermocatellispora, and Thermopolyspora.

In further embodiments, isolated microbial species may be selected fromgenera of family Leuconostocaceae, including Fructobacillus,Leuconostoc, Oenococcus, and Weissella.

In further embodiments, isolated microbial species may be selected fromgenera of family Microbacteriaceae, including Agreia, Agrococcus,Agromyces, Alpinomonas, Amnibacterium, Aureobacterium, Chryseoglobus,Clavibacter, Compostimonas, Cryobacterium, Curtobacterium,Diaminobutyricimonas, Frigoribacterium, Frondihabitans, Glacibacter,Gryllotalpicola, Gulosibacter, Herbiconiux, Homoserinimonas, Humibacter,Klugiella, Labedella, Leifsonia, Leucobacter, Lysinimonas,Marisediminicola, Microbacterium, Microcella, Microterricola,Mycetocola, Okibacterium, Phycicola, Plantibacter, Pontimonas,Pseudoclavibacter, Rathayibacter, Rhodoglobus, Salinibacterium,Schumanella, Subtercola, Yonghaparkia, and Zimmermannella.

In further embodiments, isolated microbial species may be selected fromgenera of family Micromonosporaceae, including Actinaurispora,Actinocatenispora, Actinoplanes, Allocatelliglobosispora,Amorphosporangium, Ampullariella, Asanoa, Catelliglobosispora,Catenuloplanes, Couchioplanes, Dactylosporangium, Hamadaea, Jishengella,Krasilnikovia, Longispora, Luedemannella, Micromonospora, Phytohabitans,Phytomonospora, Pilimelia, Planopolyspora, Planosporangium,Plantactinospora, Polymorphospora, Pseudosporangium, Rugosimonospora,Salinispora, Spirilliplanes, Verrucosispora, Virgisporangium, andXiangella.

In further embodiments, isolated microbial species may be selected fromgenera of family Clostridiaceae, including Acetanaerobacterium,Acetivibrio, Acidaminobacter, Alkaliphilus, Anaerobacter, Anaerostipes,Anaerotruncus, Anoxynatronum, Bryantella, Butyricicoccus,Caldanaerocella, Caloramator, Caloranaerobacter, Caminicella, CandidatusArthromitus, Clostridium, Coprobacillus, Ethanologenbacterium,Faecalibacterium, Garciella, Guggenheimella, Hespellia, Linmingia,Natronincola, Oxobacter, Parasporobacterium, Sarcina, Soehngenia,Sporobacter, Subdoligranulum, Tepidibacter, Tepidimicrobium,Thermobrachium, Thermohalobacter, and Tindallia.

In further embodiments, isolated microbial species may be selected fromgenera of family Pseudomonadaceae, including Acomonas, Azomonotrichon,Azorhizophilus, Azotobacter, Chryseomonas, Flavimonas, Mesophilobacter,Permianibacter, Pseudomonas, Rhizobacter, Rugamonas, Serpens, andThiopseudomonas.

In further embodiments, isolated microbial species may be selected fromgenera of family Nectriaceae, including Albonectria, Allonectella,Calonectria, Calostilbe, Calostilbella, Campylocarpon, Corallomycetella,Cosmospora, Cylindrocarpon, Cylindrocladiella, Cylindrocladium,Fusarium, Fusicolla, Gibberella, Glionectria, Haematonectria,Lanatonectria, Leuconectria, Nectria, Necticladiella, Neocosmoprora,Neonectria, Ophionectria, Pleogibberella, Pseudonectria, Rubrinectria,Stalagmites, Tubercularia, Viridispora, Xenocalonectria, Xenonectriella,and Zythiostroma.

In some embodiments, the disclosure provides isolated microbial speciesbelonging to genera of: Hypocreaceae, including Aphysiostroma,Cladobotryum, Gliocladium, Hypocrea, Hypocreopsis, Hypomyces, Mycogone,Nectria, Podostroma, Protocrea, Rogersonia, Sarawakus, Sepedonium,Sphaerostilbella, Sporophagomyces, Stephanoma, and Trichoderma.

In some embodiments, one or more microbes from the taxa disclosed hereinare utilized to impart one or more beneficial properties or improvedtraits to poultry production.

Furthermore, the disclosure relates to microbes having characteristicssubstantially similar to that of a microbe identified in Table 1 and/orTable 3.

The isolated microbial species, and novel strains of said species,identified in the present disclosure, are able to impart beneficialproperties or traits to poultry production.

For instance, the isolated microbes described in Table 1 and Table 3, orbioensembles of said microbes, are able to increase feed efficiency. Theincrease can be quantitatively measured, for example, by measuring theeffect that said microbial application has upon the modulation of feedefficiency. In some embodiments, feed efficiency is represented by thefeed conversion ratio, which is calculated by measuring desirable animaloutput produced per pound of feed consumed. With regard to poultry, thedesirable output is typically pounds of meat produced per pound of feedconsumed.

In some embodiments, the isolated microbial strains are microbes of thepresent disclosure that have been genetically modified. In someembodiments, the genetically modified or recombinant microbes comprisepolynucleotide sequences which do not naturally occur in said microbes.In some embodiments, the microbes may comprise heterologouspolynucleotides. In further embodiments, the heterologouspolynucleotides may be operably linked to one or more polynucleotidesnative to the microbes. In some embodiments, the isolated microbialstrains of the present disclosure further encompass mutants thereof. Insome embodiments, the present disclosure further contemplates microbialstrains having all of the identifying characteristics of the presentlydisclosed microbial strains.

In some embodiments, the heterologous polynucleotides may be reportergenes or selectable markers. In some embodiments, reporter genes may beselected from any of the family of fluorescence proteins (e.g., GFP,RFP, YFP, and the like), β-galactosidase, luciferase. In someembodiments, selectable markers may be selected from neomycinphosphotransferase, hygromycin phosphotransferase, aminoglycosideadenyltransferase, dihydrofolate reductase, acetolactase synthase,bromoxynil nitrilase, β-glucuronidase, dihydrogolate reductase, andchloramphenicol acetyltransferase. In some embodiments, the heterologouspolynucleotide may be operably linked to one or more promoter.

In some embodiments the isolated microbial strains express transgenic ornative enzymes selected from cellulases (endocellulases, exocellulases,glucosidases), pectinases, amylases, amylopectinases, ligninases, andphytases

In some embodiments, the species of the taxa provided in Table 4 are notknown to have been utilized in compositions for administration toanimals.

TABLE 4 Taxa (largely Genera) of the present disclosure not known tohave been utilized in animal agriculture. Corynebacterium VerrucosisporaClostridium XlVa Clostridium Clostridium XI Blautia FaecalibacteriumPseudomonas Hydrogenoanaerobacterium Sporobacter AcrocarposporaClostridium III Subdoligranulum Paracoccus Leuconostoc CellulosilyticumLachnospiracea Ruminococcus Anaerofilum Roseburia MicrobacteriumClostridium XlVb Verrucosispora Bacteroides

Microbial Bioensembles

In some aspects, the disclosure provides microbial bioensemblescomprising a combination of at least any two microbes selected fromamongst the microbes identified in Table 1 and Table 3.

In certain embodiments, the bioensembles of the present disclosurecomprise two microbes, or three microbes, or four microbes, or fivemicrobes, or six microbes, or seven microbes, or eight microbes, or ninemicrobes, or ten or more microbes. Said microbes of the bioensembles aredifferent microbial species, or different strains of a microbialspecies.

In some embodiments, the disclosure provides bioensembles, comprising:at least one or at least two isolated microbial species belonging togenera of: Lactobacillus, Clostridium, Faecalibacter,Hydrogenoanaerobacterium, Acrocarpospora, Bacillus, Subdoligranulum,Leuconostoc, Lachnospiracea, Anaerofilum, Microbacterium,Verrucosispora, Anaerofilum, Blautia, Pseudomonas, Sporobacter,Corynebacterium, Streptococcus, Paracoccus, Cellulosilyticum,Ruminococcus, Rosebura, Bacteroides, Filobasidium, Gibberella,Alatospora, Pichia, and Candida. Particular novel strains of species ofthese aforementioned genera can be found in Table 1 and Table 3.

In some embodiments, the disclosure provides bioensembles, comprising:at least one or at least two isolated microbial species belonging to thefamily of: Lactobacillaceae, Lachnospiraceae, Ruminococcaceae,Peptostreptococcaceae, Streptosporangiaceae, Leuconostocaceae,Microbacteriaceae, Micromonosporaceae, Clostridiaceae, Pseudomonadales,Nectriaceae, and Hypocreaceae; wherein Lachnospiraceae can be furtherspecific to Clostridium clusters XIVa and XIVb; and whereinPeptostreptococcaceae can be further specific to Clostridium cluster XI.Particular novel strains of species of these aforementioned genera canbe found in Table 1 and Table 3.

In particular aspects, the disclosure provides microbial bioensembles,comprising species as grouped in Tables 5-11. With respect to Tables5-11, the letters A through I represent a non-limiting selection ofmicrobes of the present disclosure, defined as:

-   -   A=Strain designation Ascusbbr_578 identified in Table 1;    -   B=Strain designation Ascusbbr_1436 identified in Table 1;    -   C=Strain designation Ascusbbr_33B identified in Table 1;    -   D=Strain designation Ascusbbr_409 identified in Table 1;    -   E=Strain designation Ascusbbr_185064 identified in Table 1;    -   F=Strain designation Ascusbbr_5796 identified in Table 1;    -   G=Strain designation Ascusbbr_105932 identified in Table 1;    -   H=Strain designation Ascusbbr_4729 identified in Table 1; and    -   I=Strain designation Ascusbbr_2676 identified in Table 1.

TABLE 5 Eight and Nine Strain Microbial Ensembles A, B, C, D, E, F, G, HA, B, C, D, E, F, G, I A, B, C, D, E, F, H, I A, B, C, D, E, G, H, I A,B, C, D, F, G, H, I A, B, C, E, F, G, H, I A, B, D, E, F, G, H, I A, C,D, E, F, G, H, I B, C, D, E, F, G, H, I A, B, C, D, E, F, G, H, I

TABLE 6 Seven Strain Microbial Ensembles A, B, C, D, E, F, G A, B, C, D,E, F, H A, B, C, D, E, F, I A, B, C, D, E, G, H A, B, C, D, E, G, I A,B, C, D, E, H, I A, B, C, D, F, G, H A, B, C, D, F, G, I A, B, C, D, F,H, I A, B, C, D, G, H, I A, B, C, E, F, G, H A, B, C, E, F, G, I A, B,C, E, F, H, I A, B, C, E, G, H, I A, B, C, F, G, H, I A, B, D, E, F, G,H A, B, D, E, F, G, I A, B, D, E, F, H, I A, B, D, E, G, H, I A, B, D,F, G, H, I A, B, E, F, G, H, I A, C, D, E, F, G, H A, C, D, E, F, G, IA, C, D, E, F, H, I A, C, D, E, G, H, I A, C, D, F, G, H, I A, C, E, F,G, H, I A, D, E, F, G, H, I B, C, D, E, F, G, H B, C, D, E, F, G, I B,C, D, E, F, H, I B, C, D, E, G, H, I B, C, D, F, G, H, I B, C, E, F, G,H, I B, D, E, F, G, H, I C, D, E, F, G, H, I

TABLE 7 Six Strain Bioensembles A, B, C, D, E, F A, B, C, D, E, G A, B,C, D, E, H A, B, C, D, E, I A, B, C, D, F, G A, B, C, D, F, H A, B, C,D, F, I A, B, C, D, G, H A, B, C, D, G, I A, B, C, D, H, I A, B, C, E,F, G A, B, C, E, F, H A, B, C, E, F, I A, B, C, E, G, H A, B, C, E, G, IA, B, C, E, H, I A, B, C, F, G, H A, B, C, F, G, I A, B, C, F, H, I A,B, C, G, H, I A, B, D, E, F, G A, B, D, E, F, H A, B, D, E, F, I A, B,D, E, G, H A, B, D, E, G, I A, B, D, E, H, I A, B, D, F, G, H A, B, D,F, G, I D, E, F, G, H, I C, E, F, G, H, I A, B, D, F, H, I A, B, D, G,H, I A, B, E, F, G, H A, B, E, F, G, I A, B, E, F, H, I A, B, E, G, H, IA, B, F, G, H, I A, C, D, E, F, G A, C, D, E, F, H A, C, D, E, F, I A,C, D, E, G, H A, C, D, E, G, I A, C, D, E, H, I A, C, D, F, G, H A, C,D, F, G, I A, C, D, F, H, I A, C, D, G, H, I A, C, E, F, G, H A, C, E,F, G, I A, C, E, F, H, I A, C, E, G, H, I A, C, F, G, H, I A, D, E, F,G, H A, D, E, F, G, I A, D, E, F, H, I A, D, E, G, H, I A, D, F, G, H, IA, E, F, G, H, I B, C, D, E, F, G B, C, D, E, F, H B, C, D, E, F, I B,C, D, E, G, H B, C, D, E, G, I B, C, D, E, H, I B, C, D, F, G, H B, C,D, F, G, I B, C, D, F, H, I B, C, D, G, H, I B, C, E, F, G, H B, C, E,F, G, I B, C, E, F, H, I B, C, E, G, H, I B, C, F, G, H, I B, D, E, F,G, H B, D, E, F, G, I B, D, E, F, H, I B, D, E, G, H, I B, D, F, G, H, IB, E, F, G, H, I C, D, E, F, G, H C, D, E, F, G, I C, D, E, F, H, I C,D, E, G, H, I C, D, F, G, H, I

TABLE 8 Five Strain Bioensembles A, B, C, D, E A, B, C, D, F A, B, C, D,G A, B, C, D, H A, B, C, D, I A, B, C, E, F A, B, C, E, G A, B, C, E, HA, B, C, F, H A, B, C, F, G A, B, C, F, I A, B, C, G, H A, B, C, G, I A,B, C, H, I A, B, D, E, F A, B, D, E, G A, B, D, E, I A, B, D, F, G A, B,D, F, H A, B, D, F, I A, B, D, G, H A, B, D, G, I A, B, D, H, I A, B, E,F, G A, B, E, F, I A, B, E, G, H A, B, E, G, I A, B, E, H, I A, B, F, G,H A, B, F, G, I A, B, F, H, I A, B, G, H, I A, C, D, E, G A, C, D, E, HA, C, D, E, I A, C, D, F, G A, C, D, F, H A, C, D, F, I A, C, D, G, H A,C, D, G, I A, C, E, F, G A, C, E, F, H A, C, E, F, I A, C, E, G, H A, C,E, G, I A, C, E, H, I A, C, F, G, H A, C, F, G, I A, C, G, H, I A, D, E,F, G A, D, E, F, H A, D, E, F, I A, D, E, G, H A, D, E, G, I A, D, E, H,I A, D, F, G, H A, D, F, H, I A, D, G, H, I A, E, F, G, H A, E, F, G, IA, E, F, H, I A, E, G, H, I A, F, G, H, I B, C, D, E, F B, C, D, E, H B,C, D, E, I B, C, D, F, G B, C, D, F, H B, C, D, F, I B, C, D, G, H B, C,D, G, I B, C, D, H, I B, C, E, F, H B, C, E, F, I B, C, E, G, H B, C, E,G, I B, C, E, H, I B, C, F, G, H B, C, F, G, I B, C, F, H, I B, D, E, F,G B, D, E, F, H B, D, E, F, I B, D, E, G, H B, D, E, G, I B, D, E, H, IB, D, F, G, H B, D, F, G, I B, D, G, H, I B, E, F, G, H B, E, F, G, I B,E, F, H, I B, E, G, H, I B, F, G, H, I C, D, E, F, G C, D, E, F, H C, D,E, G, H C, D, E, G, I C, D, E, H, I C, D, F, G, H C, D, F, G, I C, D, F,H, I C, D, G, H, I C, E, F, G, H C, E, F, H, I C, E, G, H, I C, F, G, H,I D, E, F, G, H D, E, F, G, I D, E, F, H, I D, E, G, H, I D, F, G, H, IA, B, C, E, I A, B, D, E, H A, B, E, F, H A, C, D, E, F A, C, D, H, I A,C, F, H, I A, D, F, G, I B, C, D, E, G B, C, E, F, G B, C, G, H, I B, D,F, H, I C, D, E, F, I C, E, F, G, I E, F, G, H, I

TABLE 9 Four Strain Bioensembles A, B, C, D A, B, C, E A, B, C, F A, B,C, G A, B, C, H A, B, C, I A, B, D, E A, B, D, F D, G, H, I A, B, D, GA, B, D, H A, B, D, I A, B, E, F A, B, E, G A, B, E, H A, B, E, I A, B,F, G E, F, G, H A, B, F, H A, D, F, H A, D, F, I A, D, G, H A, D, G, IA, D, H, I A, E, F, G A, E, F, H E, F, G, I A, B, F, I A, B, G, H A, B,G, I A, B, H, I A, C, D, E A, C, D, F A, C, D, G A, C, D, H E, F, H, IA, C, D, I A, C, E, F A, C, E, G A, C, E, H A, C, E, I A, C, F, G A, C,F, H A, C, F, I E, G, H, I A, C, G, H A, C, G, I A, C, H, I A, D, E, FA, D, E, G A, D, E, H A, D, E, I A, D, F, G F, G, H, I A, E, F, I A, E,G, H A, E, G, I A, E, H, I A, F, G, H A, F, G, I A, F, H, I A, G, H, ID, E, F, H B, C, D, E B, C, D, F B, C, D, G B, C, D, H B, C, D, I B, C,E, F B, C, E, G B, C, E, H D, E, F, I B, C, E, I B, C, F, G B, C, F, HB, C, F, I B, C, G, H B, C, G, I B, C, H, I B, D, E, F D, E, G, H B, D,E, G B, D, E, H B, D, E, I B, D, F, G B, D, F, H B, D, F, I B, D, G, HB, D, G, I D, E, G, I B, D, H, I B, E, F, G B, E, F, H B, E, F, I B, E,G, H B, E, G, I B, E, H, I B, F, G, H D, E, H, I B, F, G, I B, F, H, IB, G, H, I C, D, E, F C, D, E, G C, D, E, H C, D, E, I C, D, F, G D, F,G, H C, D, F, H C, D, F, I C, D, G, H C, D, G, I C, D, H, I C, E, F, GC, E, F, H C, E, F, I D, F, G, I C, E, G, H C, E, G, I C, E, H, I C, F,G, H C, F, G, I C, F, H, I C, G, H, I D, E, F, G D, F, H, I

TABLE 10 Three Strain Bioensembles A, B, C A, B, D A, B, E A, B, F A, B,G A, B, H A, B, I A, C, D A, C, E G, H, I E, F, H A, C, F A, C, G A, C,H A, C, I A, D, E A, D, F A, D, G A, D, H A, D, I F, H, I E, F, G A, E,F A, E, G A, E, H A, E, I A, F, G A, F, H A, F, I A, G, H A, G, I F, G,I D, H, I A, H, I B, C, D B, C, E B, C, F B, C, G B, C, H B, C, I B, D,E B, D, F F, G, H D, G, I B, D, G B, D, H B, D, I B, E, F B, E, G B, E,H B, E, I B, F, G B, F, H E, H, I E, F, I B, F, I B, G, H B, G, I B, H,I C, D, E C, D, F C, D, G C, D, H C, D, I E, G, I D, G, H C, E, F C, E,G C, E, H C, E, I C, F, G C, F, H C, F, I C, G, H C, G, I E, G, H D, F,I C, H, I D, E, F D, E, G D, E, H D, E, I D, F, G D, F, H

TABLE 11 Two Strain Bioensembles A, B A, C A, D A, E A, F A, G A, H A, IB, C B, D B, E B, F B, G B, H B, I C, D C, E C, F C, G C, H C, I D, E D,F D, G D, H D, I E, F E, G E, H E, I F, G F, H F, I G, H G, I H, I

In some embodiments, the microbial bioensembles may be selected from anymember group from Tables 5-11.

Isolated Microbes—Source Material

The microbes of the present disclosure were obtained, among otherplaces, at various locales in the United States from thegastrointestinal tract of poultry.

Isolated Microbes—Microbial Culture Techniques

The microbes of Table 1 and Table 3 were matched to their nearesttaxonomic groups by utilizing classification tools of the RibosomalDatabase Project (RDP) for 16s rRNA sequences and the User-friendlyNordic ITS Ectomycorrhiza (UNITE) database for ITS rRNA sequences.Examples of matching microbes to their nearest taxa may be found in Lanet al. (2012. PLOS one. 7(3):e32491), Schloss and Westcott (2011. Appl.Environ. Microbiol. 77(10):3219-3226), and Koljalg et al. (2005. NewPhytologist. 166(3):1063-1068).

The isolation, identification, and culturing of the microbes of thepresent disclosure can be effected using standard microbiologicaltechniques. Examples of such techniques may be found in Gerhardt, P.(ed.) Methods for General and Molecular Microbiology. American Societyfor Microbiology, Washington, D.C. (1994) and Lennette, E. H. (ed.)Manual of Clinical Microbiology, Third Edition. American Society forMicrobiology, Washington, D.C. (1980), each of which is incorporated byreference.

Isolation can be effected by streaking the specimen on a solid medium(e.g., nutrient agar plates) to obtain a single colony, which ischaracterized by the phenotypic traits described hereinabove (e.g., Grampositive/negative, capable of forming spores aerobically/anaerobically,cellular morphology, carbon source metabolism, acid/base production,enzyme secretion, metabolic secretions, etc.) and to reduce thelikelihood of working with a culture which has become contaminated.

For example, for microbes of the disclosure, biologically pure isolatescan be obtained through repeated subculture of biological samples, eachsubculture followed by streaking onto solid media to obtain individualcolonies or colony forming units. Methods of preparing, thawing, andgrowing lyophilized bacteria are commonly known, for example, Gherna, R.L. and C. A. Reddy. 2007. Culture Preservation, p 1019-1033. In C. A.Reddy, T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt, andL. R. Snyder, eds. American Society for Microbiology, Washington, D.C.,1033 pages; herein incorporated by reference. Thus freeze dried liquidformulations and cultures stored long term at −70° C. in solutionscontaining glycerol are contemplated for use in providing formulationsof the present disclosure.

The microbes of the disclosure can be propagated in a liquid mediumunder aerobic conditions, or alternatively anaerobic conditions. Mediumfor growing the bacterial strains of the present disclosure includes acarbon source, a nitrogen source, and inorganic salts, as well asspecially required substances such as vitamins, amino acids, nucleicacids and the like. Examples of suitable carbon sources which can beused for growing the microbes include, but are not limited to, starch,peptone, yeast extract, amino acids, sugars such as glucose, arabinose,mannose, glucosamine, maltose, and the like; salts of organic acids suchas acetic acid, fumaric acid, adipic acid, propionic acid, citric acid,gluconic acid, malic acid, pyruvic acid, malonic acid and the like;alcohols such as ethanol and glycerol and the like; oil or fat such assoybean oil, rice bran oil, olive oil, corn oil, sesame oil. The amountof the carbon source added varies according to the kind of carbon sourceand is typically between 1 to 100 gram(s) per liter of medium.Preferably, glucose, starch, and/or peptone is contained in the mediumas a major carbon source, at a concentration of 0.1-5% (W/V). Examplesof suitable nitrogen sources which can be used for growing the bacterialstrains of the present disclosure include, but are not limited to, aminoacids, yeast extract, tryptone, beef extract, peptone, potassiumnitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammoniumphosphate, ammonia or combinations thereof. The amount of nitrogensource varies according to the type of nitrogen source, typicallybetween 0.1 to 30 grams per liter of media. The inorganic salts,potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodiumhydrogen phosphate, magnesium sulfate, magnesium chloride, ferricsulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganoussulfate, manganous chloride, zinc sulfate, zinc chloride, cupricsulfate, calcium chloride, sodium chloride, calcium carbonate, sodiumcarbonate can be used alone or in combination. The amount of inorganicacid varies according to the kind of the inorganic salt, typicallybetween 0.001 to 10 grams per liter of medium. Examples of speciallyrequired substances include, but are not limited to, vitamins, nucleicacids, yeast extract, peptone, meat extract, malt extract, dried yeastand combinations thereof. Cultivation can be effected at a temperature,which allows the growth of the microbial strains, essentially, between20° C. and 46° C. In some aspects, a temperature range is 30° C.-39° C.For optimal growth, in some embodiments, the medium can be adjusted topH 6.0-7.4. It will be appreciated that commercially available media mayalso be used to culture the microbial strains, such as Nutrient Broth orNutrient Agar available from Difco, Detroit, MI. It will be appreciatedthat cultivation time may differ depending on the type of culture mediumused and the concentration of sugar as a major carbon source.

In some aspects, cultivation lasts between 24-96 hours. Microbial cellsthus obtained are isolated using methods, which are well known in theart. Examples include, but are not limited to, membrane filtration andcentrifugal separation. The pH may be adjusted using sodium hydroxideand the like and the culture may be dried using a freeze dryer, untilthe water content becomes equal to 4% or less. Microbial co-cultures maybe obtained by propagating each strain as described hereinabove. In someaspects, microbial multi-strain cultures may be obtained by propagatingtwo or more of the strains described hereinabove. It will be appreciatedthat the microbial strains may be cultured together when compatibleculture conditions can be employed.

Isolated Microbes—Microbial Strains

Microbes can be distinguished into a genus based on polyphasic taxonomy,which incorporates all available phenotypic and genotypic data into aconsensus classification (Vandamme et al. 1996. Polyphasic taxonomy, aconsensus approach to bacterial systematics. Microbiol Rev 1996,60:407-438). One accepted genotypic method for defining species is basedon overall genomic relatedness, such that strains which shareapproximately 70% or more relatedness using DNA-DNA hybridization, with5° C. or less ΔT_(m) (the difference in the melting temperature betweenhomologous and heterologous hybrids), under standard conditions, areconsidered to be members of the same species. Thus, populations thatshare greater than the aforementioned 70% threshold can be considered tobe variants of the same species. Another accepted genotypic method fordefining species is to isolate marker genes of the present disclosure,sequence these genes, and align these sequenced genes from multipleisolates or variants. The microbes are interpreted as belonging to thesame species if one or more of the sequenced genes share at least 97%sequence identity.

The 16S or 18S rRNA sequences or ITS sequences are often used for makingdistinctions between species and strains, in that if one of theaforementioned sequences shares less than a specified % sequenceidentity from a reference sequence, then the two organisms from whichthe sequences were obtained are said to be of different species orstrains.

Thus, one could consider microbes to be of the same species, if theyshare at least 94.5%, 95%, 97%, 98%, or 99% sequence identity across the16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.

Further, one could define microbial strains of a species, as those thatshare at least 94.5%, 95%, 97%, 98%, or 99% sequence identity across the16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.

Sequence identifiers of the present disclosure consist of SEQ IDNOs:1-387. SEQ ID NOs:1-50 and 59-387 are bacterial polynucleotidesequences encoding 16S rRNA. SEQ ID NOs:51-58 are fungal polynucleotidesequences encoding ITS sequences.

In one embodiment, microbial strains of the present disclosure includethose that comprise polynucleotide sequences that share at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any oneof SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 39, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361,362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375,376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, and 387. In afurther embodiment, microbial strains of the present disclosure includethose that comprise polynucleotide sequences that share at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any oneof SEQ ID NOs:1-387.

Comparisons may also be made with 23S rRNA sequences against referencesequences.

Unculturable microbes often cannot be assigned to a definite species inthe absence of a phenotype determination, the microbes can be given acandidatus designation within a genus provided their 16S or 18S rRNAsequences or ITS sequences subscribes to the principles of identity withknown species.

One approach is to observe the distribution of a large number of strainsof closely related species in sequence space and to identify clusters ofstrains that are well resolved from other clusters. This approach hasbeen developed by using the concatenated sequences of multiple core(house-keeping) genes to assess clustering patterns, and has been calledmultilocus sequence analysis (MLSA) or multilocus sequence phylogeneticanalysis. MLSA has been used successfully to explore clustering patternsamong large numbers of strains assigned to very closely related speciesby current taxonomic methods, to look at the relationships between smallnumbers of strains within a genus, or within a broader taxonomicgrouping, and to address specific taxonomic questions. More generally,the method can be used to ask whether bacterial species exist—that is,to observe whether large populations of similar strains invariably fallinto well-resolved clusters, or whether in some cases there is a geneticcontinuum in which clear separation into clusters is not observed.

In order to more accurately make a determination of genera, adetermination of phenotypic traits, such as morphological, biochemical,and physiological characteristics are made for comparison with areference genus archetype. The colony morphology can include color,shape, pigmentation, production of slime, etc. Features of the cell aredescribed as to shape, size, Gram reaction, extracellular material,presence of endospores, flagella presence and location, motility, andinclusion bodies. Biochemical and physiological features describe growthof the organism at different ranges of temperature, pH, salinity andatmospheric conditions, growth in presence of different sole carbon andnitrogen sources. One of ordinary skill in the art would be reasonablyapprised as to the phenotypic traits that define the genera of thepresent disclosure.

In one embodiment, the microbes taught herein were identified utilizing16S rRNA gene sequences and ITS sequences. It is known in the art that16S rRNA contains hypervariable regions that can providespecies/strain-specific signature sequences useful for bacterialidentification, and that ITS sequences can also providespecies/strain-specific signature sequences useful for fungalidentification.

Phylogenetic analysis using the rRNA genes and/or ITS sequences are usedto define “substantially similar” species belonging to common genera andalso to define “substantially similar” strains of a given taxonomicspecies. Furthermore, physiological and/or biochemical properties of theisolates can be utilized to highlight both minor and significantdifferences between strains that could lead to advantageous behavior inpoultry.

Compositions of the present disclosure may include combinations offungal spores and bacterial spores, fungal spores and bacterialvegetative cells, fungal vegetative cells and bacterial spores, fungalvegetative cells and bacterial vegetative cells, fungal vegetative cellsand fungal spores, and bacterial vegetative cells and bacterial spores.In some embodiments, compositions of the present disclosure comprisebacteria only in the form of spores. In some embodiments, compositionsof the present disclosure comprise bacteria only in the form ofvegetative cells. In some embodiments, compositions of the presentdisclosure comprise fungi only in the form of spores. In someembodiments, compositions of the present disclosure comprise fungi onlyin the form of vegetative cells. In some embodiments, compositions ofthe present disclosure comprise bacteria in the absence of fungi. Insome embodiments, compositions of the present disclosure comprise fungiin the absence of bacteria. In some embodiments, compositions of thepresent disclosure comprise VBNC bacteria and/or fungi. In someembodiments, compositions of the present disclosure include dormantbacteria and/or fungi.

Bacterial spores may include endospores and akinetes. Fungal spores mayinclude statismospores, ballistospores, autospores, aplanospores,zoospores, mitospores, megaspores, microspores, meiospores,chlamydospores, urediniospores, teliospores, oospores, carpospores,tetraspores, sporangiospores, zygospores, basidiospores, ascospores, andasciospores.

In some embodiments, spores of the composition germinate uponadministration to animals of the present disclosure. In someembodiments, spores of the composition germinate only uponadministration to animals of the present disclosure.

Microbial Compositions

In some embodiments, the microbes of the disclosure are combined intomicrobial compositions.

In some embodiments, the microbial compositions include poultry feed,such as cereals (barley, maize, oats, and the like); starches (tapiocaand the like); oilseed cakes; and vegetable wastes. In some embodiments,the microbial compositions include vitamins, minerals, trace elements,emulsifiers, aromatizing products, binders, colorants, odorants,thickening agents, and the like. In some embodiments, the microbialcompositions include one or more of an ionophore; vaccine; antibiotic;antihelmintic; virucide; nematicide; amino acids such as methionine,glycine, and arginine; fish oil; oregano; prebiotics; and biologicallyactive molecules such as enzymes.

In some embodiments, the microbial compositions of the presentdisclosure are solid. Where solid compositions are used, it may bedesired to include one or more carrier materials including, but notlimited to: mineral earths such as silicas, talc, kaolin, limestone,chalk, clay, dolomite, diatomaceous earth; calcium sulfate; magnesiumsulfate; magnesium oxide; zeolites, calcium carbonate; magnesiumcarbonate; trehalose; chitosan; shellac; albumins; starch; skim-milkpowder; sweet-whey powder; maltodextrin; lactose; inulin; dextrose;products of vegetable origin such as cereal meals, tree bark meal, woodmeal, and nutshell meal; products comprising typical poultry food stuffssuch as ground corn, barley, oats, and the like.

In some embodiments, the microbial compositions of the presentdisclosure are liquid. In further embodiments, the liquid comprises asolvent that may include water or an alcohol or a saline or carbohydratesolution, and other animal-safe solvents. In some embodiments, themicrobial compositions of the present disclosure include binders such asanimal-safe polymers, carboxymethylcellulose, starch, polyvinyl alcohol,and the like.

In some embodiments, the microbial compositions of the presentdisclosure comprise thickening agents or gelling agents such as silica,clay, natural extracts of seeds or seaweed, synthetic derivatives ofcellulose, guar gum, locust bean gum, alginates, and methylcelluloses.In some embodiments, the microbial compositions comprise anti-settlingagents such as modified starches, polyvinyl alcohol, xanthan gum, andthe like.

In some embodiments, the microbial compositions of the presentdisclosure comprise colorants including organic chromophores classifiedas nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine,anthraquinone, azine, diphenylmethane, indamine, indophenol, methine,oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene.In some embodiments, the microbial compositions of the presentdisclosure comprise trace nutrients such as salts of iron, manganese,boron, copper, cobalt, molybdenum and zinc. In some embodiments, themicrobial compositions comprise dyes, both natural and artificial. Insome embodiments, the dye is green in color. In some embodiments, thedye is red in color.

In some embodiments, the microbial compositions of the presentdisclosure comprise an animal-safe virucide, bacteriocide, ornematicide.

In some embodiments, microbial compositions of the present disclosurecomprise saccharides (e.g., monosaccharides, disaccharides,trisaccharides, polysaccharides, oligosaccharides, and the like),polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides,proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acidpolymers, silica, inorganic salts and combinations thereof. In a furtherembodiment, microbial compositions comprise polymers of agar, agarose,gelrite, and gellan gum, and the like. In some embodiments, microbialcompositions comprise plastic capsules, emulsions (e.g., water and oil),membranes, and artificial membranes. In some embodiments, emulsions orlinked polymer solutions may comprise microbial compositions of thepresent disclosure. See Harel and Bennett (U.S. Pat. No. 8,460,726B2).In one embodiment, the microbial composition comprises glucose. In oneembodiment, formulations of the microbial composition comprise glucose.

In some embodiments, microbial compositions of the present disclosurecomprise one or more oxygen scavengers, denitrifiers, nitrifiers, heavymetal chelators, and/or dechlorinators; and combinations thereof. In oneembodiment, the one or more oxygen scavengers, denitrifiers, nitrifiers,heavy metal chelators, and/or dechlorinators are not chemically activeonce the microbial compositions are mixed with food and/or water to beadministered to the poultry. In one embodiment, the one or more oxygenscavengers, denitrifiers, nitrifiers, heavy metal chelators, and/ordechlorinators are not chemically active when administered to thepoultry.

In some embodiments, microbial compositions of the present disclosureoccur in a solid form (e.g., dispersed lyophilized spores) or a liquidform (microbes interspersed in a storage medium). In some embodiments,microbial compositions of the present disclosure are added in dry formto a liquid to form a suspension immediately prior to administration.

In some embodiments, microbial compositions of the present disclosurecomprise one or more preservatives. The preservatives may be in liquidor gas formulations. The preservatives may be selected from one or moreof monosaccharide, disaccharide, trisaccharide, polysaccharide, aceticacid, ascorbic acid, calcium ascorbate, erythorbic acid, iso-ascorbicacid, erythrobic acid, potassium nitrate, sodium ascorbate, sodiumerythorbate, sodium iso-ascorbate, sodium nitrate, sodium nitrite,nitrogen, benzoic acid, calcium sorbate, ethyl lauroyl arginate,methyl-p-hydroxy benzoate, methyl paraben, potassium acetate, potassiumbenzoate, potassium bisulphite, potassium diacetate, potassium lactate,potassium metabisulphite, potassium sorbate, propyl-p-hydroxy benzoate,propyl paraben, sodium acetate, sodium benzoate, sodium bisulphite,sodium nitrite, sodium diacetate, sodium lactate, sodium metabisulphite,sodium salt of methyl-p-hydroxy benzoic acid, sodium salt ofpropyl-p-hydroxy benzoic acid, sodium sulphate, sodium sulfite, sodiumdithionite, sulphurous acid, calcium propionate, dimethyl dicarbonate,natamycin, potassium sorbate, potassium bisulfite, potassiummetabisulfite, propionic acid, sodium diacetate, sodium propionate,sodium sorbate, sorbic acid, ascorbic acid, ascorbyl palmitate, ascorbylstearate, butylated hydro-xyanisole, butylated hydroxytoluene (BHT),butylated hydroxyl anisole (BHA), citric acid, citric acid esters ofmono- and/or diglycerides, L-cysteine, L-cysteine hydrochloride, gumguaiacum, gum guaiac, lecithin, lecithin citrate, monoglyceride citrate,monoisopropyl citrate, propyl gallate, sodium metabisulphite, tartaricacid, tertiary butyl hydroquinone, stannous chloride, thiodipropionicacid, dilauryl thiodipropionate, distearyl thiodipropionate, ethoxyquin,sulfur dioxide, formic acid, or tocopherol(s).

In some embodiments, microbial compositions of the present disclosureinclude bacterial and/or fungal cells in spore form, vegetative cellform, dormant cell form, and/or lysed form. In one embodiment, the lysedcell form acts as a mycotoxin binder, e.g. mycotoxins binding to deadcells.

In some embodiments, the microbial compositions are shelf stable in arefrigerator (35-40° F.) for a period of at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60days. In some embodiments, the microbial compositions are shelf stablein a refrigerator (35-40° F.) for a period of at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60weeks.

In some embodiments, the microbial compositions are shelf stable at roomtemperature (68-72° F.) or between 50-77° F. for a period of at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, or 60 days. In some embodiments, the microbial compositions areshelf stable at room temperature (68-72° F.) or between 50-77° F. for aperiod of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at−23-35° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at −23-35° F.for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at77-100° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at 77-100° F.for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at101-213° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at 101-213° F.for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of about 1 to100, about 1 to 95, about 1 to 90, about 1 to 85, about 1 to 80, about 1to 75, about 1 to 70, about 1 to 65, about 1 to 60, about 1 to 55, about1 to 50, about 1 to 45, about 1 to 40, about 1 to 35, about 1 to 30,about 1 to 25, about 1 to 20, about 1 to 15, about 1 to 10, about 1 to5, about 5 to 100, about 5 to 95, about 5 to 90, about 5 to 85, about 5to 80, about 5 to 75, about 5 to 70, about 5 to 65, about 5 to 60, about5 to 55, about 5 to 50, about 5 to 45, about 5 to 40, about 5 to 35,about 5 to 30, about 5 to 25, about 5 to 20, about 5 to 15, about 5 to10, about 10 to 100, about 10 to 95, about 10 to 90, about 10 to 85,about 10 to 80, about 10 to 75, about 10 to 70, about 10 to 65, about 10to 60, about 10 to 55, about 10 to 50, about 10 to 45, about 10 to 40,about 10 to 35, about 10 to 30, about 10 to 25, about 10 to 20, about 10to 15, about 15 to 100, about 15 to 95, about 15 to 90, about 15 to 85,about 15 to 80, about 15 to 75, about 15 to 70, about 15 to 65, about 15to 60, about 15 to 55, about 15 to 50, about 15 to 45, about 15 to 40,about 15 to 35, about 15 to 30, about 15 to 25, about 15 to 20, about 20to 100, about 20 to 95, about 20 to 90, about 20 to 85, about 20 to 80,about 20 to 75, about 20 to 70, about 20 to 65, about 20 to 60, about 20to 55, about 20 to 50, about 20 to 45, about 20 to 40, about 20 to 35,about 20 to 30, about 20 to 25, about 25 to 100, about 25 to 95, about25 to 90, about 25 to 85, about 25 to 80, about 25 to 75, about 25 to70, about 25 to 65, about 25 to 60, about 25 to 55, about 25 to 50,about 25 to 45, about 25 to 40, about 25 to 35, about 25 to 30, about 30to 100, about 30 to 95, about 30 to 90, about 30 to 85, about 30 to 80,about 30 to 75, about 30 to 70, about 30 to 65, about 30 to 60, about 30to 55, about 30 to 50, about 30 to 45, about 30 to 40, about 30 to 35,about 35 to 100, about 35 to 95, about 35 to 90, about 35 to 85, about35 to 80, about 35 to 75, about 35 to 70, about 35 to 65, about 35 to60, about 35 to 55, about 35 to 50, about 35 to 45, about 35 to 40,about 40 to 100, about 40 to 95, about 40 to 90, about 40 to 85, about40 to 80, about 40 to 75, about 40 to 70, about 40 to 65, about 40 to60, about 40 to 55, about 40 to 50, about 40 to 45, about 45 to 100,about 45 to 95, about 45 to 90, about 45 to 85, about 45 to 80, about 45to 75, about 45 to 70, about 45 to 65, about 45 to 60, about 45 to 55,about 45 to 50, about 50 to 100, about 50 to 95, about 50 to 90, about50 to 85, about 50 to 80, about 50 to 75, about 50 to 70, about 50 to65, about 50 to 60, about 50 to 55, about 55 to 100, about 55 to 95,about 55 to 90, about 55 to 85, about 55 to 80, about 55 to 75, about 55to 70, about 55 to 65, about 55 to 60, about 60 to 100, about 60 to 95,about 60 to 90, about 60 to 85, about 60 to 80, about 60 to 75, about 60to 70, about 60 to 65, about 65 to 100, about 65 to 95, about 65 to 90,about 65 to 85, about 65 to 80, about 65 to 75, about 65 to 70, about 70to 100, about 70 to 95, about 70 to 90, about 70 to 85, about 70 to 80,about 70 to 75, about 75 to 100, about 75 to 95, about 75 to 90, about75 to 85, about 75 to 80, about 80 to 100, about 80 to 95, about 80 to90, about 80 to 85, about 85 to 100, about 85 to 95, about 85 to 90,about 90 to 100, about 90 to 95, or 95 to 100 weeks

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of 1 to 100, 1to 95, 1 to 90, 1 to 85, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 5 to 100, 5 to 95, 5 to 90, 5 to 85, 5 to 80, 5to 75, 5 to 70, 5 to 65, 5 to 60, 5 to 55, 5 to 50, 5 to 45, 5 to 40, 5to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 100, 10 to 95,10 to 90, 10 to 85, 10 to 80, 10 to 75, 10 to 70, 10 to 65, 10 to 60, 10to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to20, 10 to 15, 15 to 100, 15 to 95, 15 to 90, 15 to 85, 15 to 80, 15 to75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 100, 20 to 95, 20 to90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to100, 25 to 95, 25 to 90, 25 to 85, 25 to 80, 25 to 75, 25 to 70, 25 to65, 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to30, 30 to 100, 30 to 95, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to70, 30 to 65, 30 to 60, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to35, 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to100, 40 to 95, 40 to 90, 40 to 85, 40 to 80, 40 to 75, 40 to 70, 40 to65, 40 to 60, 40 to 55, 40 to 50, 40 to 45, 45 to 100, 45 to 95, 45 to90, 45 to 85, 45 to 80, 45 to 75, 45 to 70, 45 to 65, 45 to 60, 45 to55, 45 to 50, 50 to 100, 50 to 95, 50 to 90, 50 to 85, 50 to 80, 50 to75, 50 to 70, 50 to 65, 50 to 60, 50 to 55, 55 to 100, 55 to 95, 55 to90, 55 to 85, 55 to 80, 55 to 75, 55 to 70, 55 to 65, 55 to 60, 60 to100, 60 to 95, 60 to 90, 60 to 85, 60 to 80, 60 to 75, 60 to 70, 60 to65, 65 to 100, 65 to 95, 65 to 90, 65 to 85, 65 to 80, 65 to 75, 65 to70, 70 to 100, 70 to 95, 70 to 90, 70 to 85, 70 to 80, 70 to 75, 75 to100, 75 to 95, 75 to 90, 75 to 85, 75 to 80, 80 to 100, 80 to 95, 80 to90, 80 to 85, 85 to 100, 85 to 95, 85 to 90, 90 to 100, 90 to 95, or 95to 100 weeks.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of about 1 to36, about 1 to 34, about 1 to 32, about 1 to 30, about 1 to 28, about 1to 26, about 1 to 24, about 1 to 22, about 1 to 20, about 1 to 18, about1 to 16, about 1 to 14, about 1 to 12, about 1 to 10, about 1 to 8,about 1 to 6, about 1 one 4, about 1 to 2, about 4 to 36, about 4 to 34,about 4 to 32, about 4 to 30, about 4 to 28, about 4 to 26, about 4 to24, about 4 to 22, about 4 to 20, about 4 to 18, about 4 to 16, about 4to 14, about 4 to 12, about 4 to 10, about 4 to 8, about 4 to 6, about 6to 36, about 6 to 34, about 6 to 32, about 6 to 30, about 6 to 28, about6 to 26, about 6 to 24, about 6 to 22, about 6 to 20, about 6 to 18,about 6 to 16, about 6 to 14, about 6 to 12, about 6 to 10, about 6 to8, about 8 to 36, about 8 to 34, about 8 to 32, about 8 to 30, about 8to 28, about 8 to 26, about 8 to 24, about 8 to 22, about 8 to 20, about8 to 18, about 8 to 16, about 8 to 14, about 8 to 12, about 8 to 10,about 10 to 36, about 10 to 34, about 10 to 32, about 10 to 30, about 10to 28, about 10 to 26, about 10 to 24, about 10 to 22, about 10 to 20,about 10 to 18, about 10 to 16, about 10 to 14, about 10 to 12, about 12to 36, about 12 to 34, about 12 to 32, about 12 to 30, about 12 to 28,about 12 to 26, about 12 to 24, about 12 to 22, about 12 to 20, about 12to 18, about 12 to 16, about 12 to 14, about 14 to 36, about 14 to 34,about 14 to 32, about 14 to 30, about 14 to 28, about 14 to 26, about 14to 24, about 14 to 22, about 14 to 20, about 14 to 18, about 14 to 16,about 16 to 36, about 16 to 34, about 16 to 32, about 16 to 30, about 16to 28, about 16 to 26, about 16 to 24, about 16 to 22, about 16 to 20,about 16 to 18, about 18 to 36, about 18 to 34, about 18 to 32, about 18to 30, about 18 to 28, about 18 to 26, about 18 to 24, about 18 to 22,about 18 to 20, about 20 to 36, about 20 to 34, about 20 to 32, about 20to 30, about 20 to 28, about 20 to 26, about 20 to 24, about 20 to 22,about 22 to 36, about 22 to 34, about 22 to 32, about 22 to 30, about 22to 28, about 22 to 26, about 22 to 24, about 24 to 36, about 24 to 34,about 24 to 32, about 24 to 30, about 24 to 28, about 24 to 26, about 26to 36, about 26 to 34, about 26 to 32, about 26 to 30, about 26 to 28,about 28 to 36, about 28 to 34, about 28 to 32, about 28 to 30, about 30to 36, about 30 to 34, about 30 to 32, about 32 to 36, about 32 to 34,or about 34 to 36 months.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of 1 to 36 1 to34 1 to 32 1 to 30 1 to 28 1 to 26 1 to 24 1 to 22 1 to 20 1 to 18 to 161 to 14 1 to 12 1 to 10 1 to 8 1 to 6 1 one 4 1 to 2 4 to 36 4 to 34 4to 32 4 to 30 4 to 28 4 to 26 4 to 24 4 to 22 4 to 20 4 to 18 4 to 16 4to 14 4 to 12 4 to 10 4 to 8 4 to 6 6 to 36 6 to 34 6 to 32 6 to 30 6 to28 6 to 26 6 to 24 6 to 22 6 to 20 6 to 18 6 to 16 6 to 14 6 to 12 6 to10 6 to 8 8 to 36 8 to 34 8 to 32 8 to 30 8 to 28 8 to 26 8 to 24 8 to22 8 to 20 8 to 18 8 to 16 8 to 14 8 to 12 8 to 10 10 to 36 10 to 34 10to 32 10 to 30 10 to 28 10 to 26 10 to 24 10 to 22 10 to 20 10 to 18 10to 16 10 to 14 10 to 12 12 to 36 12 to 34 12 to 32 12 to 30 12 to 28 12to 26 12 to 24 12 to 22 12 to 20 12 to 18 12 to 16 12 to 14 14 to 36 14to 34 14 to 32 14 to 30 14 to 28 14 to 26 14 to 24 14 to 22 14 to 20 14to 18 14 to 16 16 to 36 16 to 34 16 to 32 16 to 30 16 to 28 16 to 26 16to 24 16 to 22 16 to 20 16 to 18 18 to 36 18 to 34 18 to 32 18 to 30 18to 28 18 to 26 18 to 24 18 to 22 18 to 20 20 to 36 20 to 34 20 to 32 20to 30 20 to 28 20 to 26 20 to 24 20 to 22 22 to 36 22 to 34 22 to 32 22to 30 22 to 28 22 to 26 22 to 24 24 to 36 24 to 34 24 to 32 24 to 30 24to 28 24 to 26 26 to 36 26 to 34 26 to 32 26 to 30 26 to 28 28 to 36 28to 34 28 to 32 28 to 30 30 to 36 30 to 34 30 to 32 32 to 36 32 to 34, orabout 34 to 36.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at any of the disclosed temperatures and/ortemperature ranges and spans of time at a relative humidity of at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, or 98%.

Moisture content is a measurement of the total amount of water in acomposition, usually expressed as a percentage of the total weight. Themoisture content is a useful measurement for determining the dry weightof a composition, and it can be used to confirm whether thedesiccation/drying process of a composition is complete. The moisturecontent is calculated by dividing the (wet weight of the compositionminus the weight after desiccating/drying) by the wet weight of thecomposition, and multiplying by 100.

Moisture content defines the amount of water in a composition, but wateractivity explains how the water in the composition will react withmicroorganisms. The greater the water activity, the fastermicroorganisms are able to grow. Water activity is calculated by findingthe ratio of the vapor pressure in a composition to the vapor pressureof pure water. More specifically, the water activity is the partialvapor pressure of water in a composition divided by the standard statepartial vapor pressure of pure water. Pure distilled water has a wateractivity of 1. A determination of water activity of a composition is notthe amount of water in a composition, rather it is the amount of excessamount of water that is available for microorganisms to use.Microorganisms have a minimal and optimal water activity for growth.

In some embodiments, the microbial compositions of the presentdisclosure are desiccated. A microbial composition is desiccated if themoisture content of the composition is between 0% and 20%.

In some embodiments, the microbial compositions of the presentdisclosure have a moisture content of about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%,about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%,about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%,about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%,about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%,about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%,about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or about 100%.

In some embodiments, the microbial compositions of the presentdisclosure have a moisture content of less than 0.5%, less than 0.6%,less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than2%, less than 3%, less than 4%, less than 5%, less than 6%, less than7%, less than 8%, less than 9%, less than 10%, less than 11%, less than12%, less than 13%, less than 14%, less than 15%, less than 16%, lessthan 17%, less than 18%, less than 19%, less than 20%, less than 21%,less than 22%, less than 23%, less than 24%, less than 25%, less than26%, less than 27%, less than 28%, less than 29%, less than 30%, lessthan 31%, less than 32%, less than 33%, less than 34%, less than 35%,less than 36%, less than 37%, less than 38%, less than 39%, less than40%, less than 41%, less than 42%, less than 43%, less than 44%, lessthan 45%, less than 46%, less than 47%, less than 48%, less than 49%,less than 50%, less than 51%, less than 52%, less than 53%, less than54%, less than 55%, less than 56%, less than 57%, less than 58%, lessthan 59%, less than 60%, less than 61%, less than 62%, less than 63%,less than 64%, less than 65%, less than 66%, less than 67%, less than68%, less than 69%, less than 70%, less than 71%, less than 72%, lessthan 73%, less than 74%, less than 75%, less than 76%, less than 77%,less than 78%, less than 79%, less than 80%, less than 81%, less than82%, less than 83%, less than 84%, less than 85%, less than 86%, lessthan 87%, less than 88%, less than 89%, less than 90%, less than 91%,less than 92%, less than 93%, less than 94%, less than 95%, less than96%, less than 97%, less than 98%, less than 99%, or less than 100%.

In some embodiments, the microbial compositions of the presentdisclosure have a moisture content of less than about 0.5%, less thanabout 0.6%, less than about 0.7%, less than about 0.8%, less than about0.9%, less than about 1%, less than about 2%, less than about 3%, lessthan about 4%, less than about 5%, less than about 6%, less than about7%, less than about 8%, less than about 9%, less than about 10%, lessthan about 11%, less than about 12%, less than about 13%, less thanabout 14%, less than about 15%, less than about 16%, less than about17%, less than about 18%, less than about 19%, less than about 20%, lessthan about 21%, less than about 22%, less than about 23%, less thanabout 24%, less than about 25%, less than about 26%, less than about27%, less than about 28%, less than about 29%, less than about 30%, lessthan about 31%, less than about 32%, less than about 33%, less thanabout 34%, less than about 35%, less than about 36%, less than about37%, less than about 38%, less than about 39%, less than about 40%, lessthan about 41%, less than about 42%, less than about 43%, less thanabout 44%, less than about 45%, less than about 46%, less than about47%, less than about 48%, less than about 49%, less than about 50%, lessthan about 51%, less than about 52%, less than about 53%, less thanabout 54%, less than about 55%, less than about 56%, less than about57%, less than about 58%, less than about 59%, less than about 60%, lessthan about 61%, less than about 62%, less than about 63%, less thanabout 64%, less than about 65%, less than about 66%, less than about67%, less than about 68%, less than about 69%, less than about 70%, lessthan about 71%, less than about 72%, less than about 73%, less thanabout 74%, less than about 75%, less than about 76%, less than about77%, less than about 78%, less than about 79%, less than about 80%, lessthan about 81%, less than about 82%, less than about 83%, less thanabout 84%, less than about 85%, less than about 86%, less than about87%, less than about 88%, less than about 89%, less than about 90%, lessthan about 91%, less than about 92%, less than about 93%, less thanabout 94%, less than about 95%, less than about 96%, less than about97%, less than about 98%, less than about 99%, or less than about 100%.

In some embodiments, the microbial compositions of the presentdisclosure have a moisture content of 1% to 100%, 1% to 95%, 1% to 90%,1% to 85%, 1% to 80%, 1% to 75%, 1% to 70%, 1% to 65%, 1% to 60%, 1% to55%, 1% to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1% to 25%,1% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 5% to 100%, 5% to 95%, 5% to90%, 5% to 85%, 5% to 80%, 5% to 75%, 5% to 70%, 5% to 65%, 5% to 60%,5% to 55%, 5% to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to25%, 5% to 20%, 5% to 15%, 5% to 10%, 10% to 100%, 10% to 95%, 10% to90%, 10% to 85%, 10% to 80%, 10% to 75%, 10% to 70%, 10% to 65%, 10% to60%, 10% to 55%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to30%, 10% to 25%, 10% to 20%, 10% to 15%, 15% to 100%, 15% to 95%, 15% to90%, 15% to 85%, 15% to 80%, 15% to 75%, 15% to 70%, 15% to 65%, 15% to60%, 15% to 55%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35%, 15% to30%, 15% to 25%, 15% to 20%, 20% to 100%, 20% to 95%, 20% to 90%, 20% to85%, 20% to 80%, 20% to 75%, 20% to 70%, 20% to 65%, 20% to 60%, 20% to55%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to25%, 25% to 100%, 25% to 95%, 25% to 90%, 25% to 85%, 25% to 80%, 25% to75%, 25% to 70%, 25% to 65%, 25% to 60%, 25% to 55%, 25% to 50%, 25% to45%, 25% to 40%, 25% to 35%, 25% to 30%, 30% to 100%, 30% to 95%, 30% to90%, 30% to 85%, 30% to 80%, 30% to 75%, 30% to 70%, 30% to 65%, 30% to60%, 30% to 55%, 30% to 50%, 30% to 45%, 30% to 40%, 30% to 35%, 35% to100%, 35% to 95%, 35% to 90%, 35% to 85%, 35% to 80%, 35% to 75%, 35% to70%, 35% to 65%, 35% to 60%, 35% to 55%, 35% to 50%, 35% to 45%, 35% to40%, 40% to 100%, 40% to 95%, 40% to 90%, 40% to 85%, 40% to 80%, 40% to75%, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 40% to 50%, 40% to45%, 45% to 100%, 45% to 95%, 45% to 90%, 45% to 85%, 45% to 80%, 45% to75%, 45% to 70%, 45% to 65%, 45% to 60%, 45% to 55%, 45% to 50%, 50% to100%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to70%, 50% to 65%, 50% to 60%, 50% to 55%, 55% to 100%, 55% to 95%, 55% to90%, 55% to 85%, 55% to 80%, 55% to 75%, 55% to 70%, 55% to 65%, 55% to60%, 60% to 100%, 60% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to75%, 60% to 70%, 60% to 65%, 65% to 100%, 65% to 95%, 65% to 90%, 65% to85%, 65% to 80%, 65% to 75%, 65% to 70%, 70% to 100%, 70% to 95%, 70% to90%, 70% to 85%, 70% to 80%, 70% to 75%, 75% to 100%, 75% to 95%, 75% to90%, 75% to 85%, 75% to 80%, 80% to 100%, 80% to 95%, 80% to 90%, 80% to85%, 85% to 100%, 85% to 95%, 85% to 90%, 90% to 100%, 90% to 95%, or95% to 100%.

Microbial compositions suitable for use in the present application aredisclosed in Embree et al. (PCT/US2017/028015).

In some embodiments, the microbial compositions of the presentdisclosure possess a water activity of at least 0.05, at least 0.075, atleast 0.1, at least 0.1.25, at least 0.15, at least 0.175, at least 0.2,at least 0.225, at least 0.25, at least 0.275, at least 0.3, at least0.325, at least 0.35, at least 0.375, at least 0.4, at least 0.425, atleast 0.45, at least 0.475, at least 0.5, at least 0.525, at least 0.55,at least 0.575, or at least 0.6.

In some embodiments, the microbial compositions of the presentdisclosure possess a water activity of less than 0.05, less than 0.075,less than 0.1, less than 0.1.25, less than 0.15, less than 0.175, lessthan 0.2, less than 0.225, less than 0.25, less than 0.275, less than0.3, less than 0.325, less than 0.35, less than 0.375, less than 0.4,less than 0.425, less than 0.45, less than 0.475, less than 0.5, lessthan 0.525, less than 0.55, less than 0.575, or less than 0.6.

Poultry

Poultry include chickens(broilers/fryers/roasters/capons/roosters/stewing hens), turkeys,grouse, New World quail, Old World quail, partridges, ptarmigans,junglefowl, peafowl, ducks, geese, swans, emus, and ostriches.

Broiler chickens of the present disclosure include: Cobb 500, Cobb 700,Cobb Avian 48, Cobb Sasso, Ross 308, Ross 708, Ross PM3, Jersey Giant,Cornish Cross, Delaware, Dorking, Buckeye, Campine, Chantecler,Crevecoeur, Holland, Modern Game, Nankin, Redcap, Russian, Orloff,Spanish, Sultan, Sumatra, Yokohama, Andalusian, Buttercup, Cubalaya,Faverolles, Java, Lakenvelder, Langshan, Malay, Phoenix, Ancona, Aseel,Brahma, Catalana, Cochin, Cornish, Dominique, Hamburg, Houdan, LaFleche, Minorca, New Hampshire, Old English Game, Polish, Rhode IslandWhite, Sebright, Shamo, Australorp, Leghorn, Orpington, Plymouth Rock,Rhode Island Red, Sussex, Wyandotte, Araucana, Iowa Blue, Lamona, ManxRumpy, Naked Neck, Asil, Kadacknath Bursa, Hubbard, Hubbard, Cobb,Hubbard, Lohman, Anak 2000, Avian-34, Starbra, Sam Rat, Bowans, Hyline,BV-300, H & N Nick, Dekalb Lohman, ILI-80, Golden-92, Priya, Sonali,Devendra, B-77, Caribro-91, Varna, Caribro naked necked, Caribromulticolored, Aviagen, Ross, Arbor Acres, Indian River, Peterson,Cobb-Vantress, Avian Sasso, Hybro, Groupe Grimaud, Grimaud Frere,Ameraucana, Silkie, Marans, Rosecomb, Welsummer, Barnevelder, Bantam,Asil, Chantecler, Croad, Houdan, Pekin, Frizzle, Serama, Orloff, Ac,Aseel, Baheij, Bandara, and hybrids thereof.

Egg-laying chickens of the present disclosure include: Ameraucana,Ancona, Andalusian, Appenzeller, Araucana, Australorp, Barnevelder,Brahma, Buckeye, Buttercup, Campine, Catalana, Chantecler, Cochin,Cornish, Crevecoeur, Cubalaya, Deleware, Dominique, Dorking, Faverolles,Fayoumi, Hamburg, Holland, Houdan, Jaerhon, Java, Jersey Giant, LaFleche, Lakenvelder, Lamona, Langsham, Leghorn, Marans, Minorca, NackedNeck, New Hampshire, Orloff, Orpington, Penedesenca, Phoenix, PlymouthRock, Polish, Redcap, Rhode Island, Spanish, Sultan, Sussex, Welsumer,Wyandotte, Yokohama, and hybrids thereof.

While distinctions are made between broiler chickens and egg-layingchickens, embodiments of the present disclosure utilize broilerchickens, egg-laying chickens, and/or multipurpose chickens.

Chickens in commercial settings have been found to exhibit a high degreeof bird-to-bird and location-to-location variability in terms of themicrobial compositions of the gastrointestinal tract. While theincreased hygiene of modern commercial hatcheries are thought to quellthe bird losses, the increased hygiene may be contributing to the lackof colonization of the chicks/birds by maternally derived bacteria. Theincreased variability of the microbial compositions of thegastrointestinal tract of the birds in these facilities may lead toincreased bird-to-bird and location-to-location variability that in turnresults in a highly variable flock of birds with considerabledifferences in health, weight, and other attributes that affectcommercial viability of the flock. The high variability amongst membersof a single flock or even differences between locations impacts thesusceptibility to pathogens, weight gain, and response to antibiotictreatment. See Stanley et al. (2013. PLOS ONE. 8(12):1-7; e84290).

In some embodiments, the administration of one or more microbes and/orbioensembles of the present disclosure early in a bird's life decreasesthe variability of the gut microbiome between birds and furtherestablishes a stable gut microbiome.

In some embodiments, the variability of the gut microbiome is measuredas the total number of species present in the gut at one or morelocations. In some embodiments, the variability of the gut microbiome ismeasured as the presence or absence of particular taxa present in thegut at one or more locations. In some embodiments, the variability ofthe gut microbiome is measured as a difference in abundance ofparticular taxa present in the gut at one or more locations.

In some embodiments, the administration of one or more microbes and/orbioensembles of the present disclosure reduces the amount of timerequired for the gut microbiome to reach a stabilized state. In someembodiments, the administration of one or more microbes and/orbioensembles of the present disclosure reduces the amount of timerequired for the gut microbiome to reach a matured state.

In some embodiments, the administration of one or more microbes and/orbioensembles of the present disclosure results in poultry of the presentdisclosure reaching a stabilized state of the gut microbiome; areduction in the variability of the gut microbiome.

In some embodiments, the stabilized state of the gut microbiome isreached when the gut microbiome of poultry contains about 10, about 20,about, 30, about 40, about 50, about 60, about 70, about 80, about 90,about 100, about 120, about 130, about 140, about 150, about 160, about170, about 180, about 190, about 200, about 250, about 300, about 400,about 500, about 600, about 700, about 800, about 900, about 1,000,about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500,about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about9,500, or about 10,000 different species.

In some embodiments, the stabilized state of the gut microbiome isreached when the gut microbiome of poultry contains between about 10 toabout 50, about 10 to about 100, about 50 to about 100, about 50 toabout 200, about 100 to about 150, about 100 to about 200, about 100 toabout 400, about 200 to about 500, about 200 to about 700, about 400 toabout 800, about 500 to about 1,000, about 500 to about 2,000, about1,000 to about 2,000, about 1,000 to about 5,000, about 5,000 to about7,000, about 5,000 to about 10,000, or about 8,000 to about 10,000different species.

In some embodiments, at least 5%, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or at least 95% of the poultry in apen/flock/hatchery reach a stabilized state after administration of oneor more microbes and/or bioensembles of the present disclosure.

I. Competitive Exclusion and Immunomodulation

The ability of avian gut pathogens to adhere to a variety of moleculesdisplayed in the glycocalyx/extracellular matrix of the avian gut isknown to contribute to the pathogenicity of those particularorganisms/strains. See Martin and Smyth (2010. Anaerobe. 16:533-539) andWade et al. (2016. Vet. Microbiol. 197:53-61). Microbes passed fromadult poultry to hatchlings aid in protecting the hatchling during avulnerable stage when the maternal antibodies and antimicrobialcompounds (lysozymes among others) received from the egg white havedepleted and the hatchling's immune system is not yet fully developed.

In some embodiments, the microbes and/or bioensembles of the presentdisclosure are producing the antimicrobial compounds. In someembodiments, the microbes and/or bioensembles are stimulating othermicrobes in the poultry to produce the antimicrobial compounds. In someembodiments, the microbes and/or bioensembles of the present disclosureare stimulating the immune system of the poultry, resulting in anincrease in the production of antimicrobial compounds. In someembodiments, the antimicrobial compounds are produced in thegastrointestinal tract of the fowl and remain localized to thegastrointestinal tract. In some embodiments the antimicrobial compoundsare produced distally from the gastrointestinal tract and localize tothe gastrointestinal tract. In some embodiments, the antimicrobialcompounds are circulated systemically in the poultry. In some aspects,the antimicrobial compounds include chemicals and compounds that areinhibitory, sporicidal, virucidal, bacteriostatic, or bacteriocidal toone or more microbes. In further embodiments, the antimicrobialcompounds include chemicals and compounds that are inhibitory,sporicidal, virucidal, bacteriostatic, or bacteriocidal to one or morepathogenic microbes. In some embodiments, the antimicrobial compoundsare as described throughout, and further including hydrogen peroxide,diacetyl, carbon dioxide, and bacteriocins (e.g., nisin, pediocin A,pediocin AcH, leucocin, helveticin J, and canobacteriocin). Theantimicrobials presented herein are presented as exemplaryantimicrobials and are not intended to limit the antimicrobialscontemplated.

In some embodiments, the microbes and/or bioensemples of the presentdisclosure are administered to mature the gut/mucosal immune system morequickly than that of poultry that have not been administered themicrobes and/or bioensembles. A mature gut/mucosal immune system is incontrast to a naïve gut/mucosal immune system, with regard to bothadaptive immunity and innate immunity.

In some embodiments, microbes and bioensembles of the present disclosureare administered to competitively exclude microbial pathogens fromcausing a disease state in the poultry.

In some embodiments, microbes and bioensembles of the present disclosurecompetitively bind molecules of the glycocalyx/extracellular matrix ofthe gut cell walls to preclude or competitively inhibit pathogens fromadhering to lectins and other molecules such as collagens (particularlytypes-III, IV, and V), gelatin, fibrinogen, laminin, and vitronectin.Pathogen adherence to these molecules are believed to contribute to thevirulence of the pathogens.

In some embodiments, administration of microbial compositions of thepresent disclosure result in a decrease in the binding of pathogenicmicrobes to the glycocalyx/extracellular matrix of the cells of thepoultry gastrointestinal tract.

In some embodiments, the microbial compositions of the presentdisclosure result in the binding of the administered microbes to theglycocalyx/extracellular matrix, preventing pathogenic microbes fromadhering to the glycocalyx/extracellular matrix and preventingpathogenic disease.

In some embodiments, the microbial compositions of the presentdisclosure result in the chemical modification of the molecules of theglycocalyx/extracellular matrix by the administered microbialcomposition, preventing pathogenic microbes from adhering to theglycocalyx/extracellular matrix and preventing pathogenic disease.

In some embodiments, the molecules bound or chemically modified by theadministered microbes are selected from lectins, collagens, gelatins,fibrinogens, laminins, and vitronectins.

In some embodiments, the gastrointestinal tract of poultry exhibit adecreased pH upon administration of one or more microbes and/orbioensembles of the present disclosure. The decreased pH may occur inthe crop, proventriculus, gizzard/ventriculus, duodenum, smallintestine, ceca, large intestine, or the cloaca.

In some embodiments, the gastrointestinal tract of poultry exhibit adecreased pH upon administration of one or more microbes and/orbioensembles of the present disclosure by at least 0.2, at least 0.4, atleast 0.6, at least 0.8, at least 1, at least 1.2, at least 1.4, atleast 1.6, at least 1.8, at least 2, at least 2.2, at least 2.4, atleast 2.6, at least 2.8, at least 3, at least 3.2, at least 3.4, atleast 3.6, at least 3.8, at least 4, at least 4.2, at least 4.4, atleast 4.6, at least 4.8, at least 5, at least 5.2, at least 5.4, atleast 5.6, at least 5.8, at least 6, at least 6.2, at least 6.4, atleast 6.6, at least 6.8, or at least 7.

In some embodiments, the decrease in pH in the gastrointestinal tract ofpoultry prevents pathogenic microbes from outcompeting thenon-pathogenic microbes in the gastrointestinal tract of poultry.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry stimulate the production of B cells. Insome embodiments, B cells are selected from regulatory B cells, B-1cells, B-2 cells, marginal zone B cells, follicular B cells, memory Bcells, plasma cells, and plasmablasts.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry result in an increase of one or more typesof B cells by at least 1%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95%.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry stimulate the production of T cells. Insome embodiments, T cells are selected from 76 (gamma delta) T cells, αβ(alpha beta) T cells, natural killer T cells, regulatory T cells, memoryT cells, cytotoxic T cells, helper T cells, and effector T cells.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry result in an increase of one or more typesof T cells by at least 1%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95%.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry result in an increase in the number ofisolated lymphoid follicles (ILFs).

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry result in an increase of isolated lymphoidfollicles by at least 1%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95%.

In some embodiments, the administration of microbial compositions of thepresent disclosure result in the modulation of the gene expression ofmucins, tight junction polypeptides, and cytokines. In some embodiments,the modulation of the gene expression of mucins, tight junctionpolypeptides, and cytokines results in an increase of the geneexpression of said molecules. In some embodiments, the modulation of thegene expression of mucins, tight junction polypeptides, and cytokinesresults in a decrease of the gene expression of said molecules.

In some embodiments, the cytokines are selected fromgranulocyte-macrophage stimulating factor (GM-CSF), IL-1, IL-12, IL-18,tumor necrosis factor (TNF), and interferon gamma (IFN-7). In someembodiments, the cytokines are selected from IL-4, IL-6, IL-10, IL-11,IL-13, and IL-1 receptor agonist.

In some embodiments, the administration of microbial compositions of thepresent disclosure result in a decrease of gut inflammation in poultry,as measured by the serum levels of inflammation markers. In someembodiment, the inflammation markers are selected from al-acidglycoprotein (AGP), IL-8, IL-1β, transforming growth factor (TGF-β4),and fatty acid-binding protein (FABP2).

In some embodiments, administration of the microbial compositions toegg-laying poultry results in an increase in the innate immune responsein the resulting eggs of the egg-laying broilers. In some aspects,administration of the microbial compositions to the eggs of egg-layingpoultry results in an increase in the innate immune response in theresulting eggs of the egg-laying poultry. In some aspects,administration of the microbial compositions to poultry results in animprovement in the innate immune response in the eggs of egg-layingpoultry. The improvement or increase is measured against eggs/poultrythat were not administered the microbial compositions. In someembodiments, the improvement or increase in the innate immune responsein the eggs results in an increased hatching success, increasedincidence of normal chick morphology, increased incidence of embryosurvival, increased growth rate and total body mass in chicks.

In some embodiments, administration of the microbial compositions toegg-laying poultry or to eggs of egg-laying poultry results in either adecrease or an increase in egg-white proteins in the eggs.

In some embodiments, the innate immune response includes an improvementin the innate immune response in eggs of egg-laying poultry, theimprovement is an increase or decrease in antimicrobials such aslysozyme, steroids, egg-white avidin, apoprotein, ovomucoid, ovomucin,ovoflavoprotein, ovoinhibitor, and conalbumin (ovotransferrin) in theegg. In some embodiments, the innate immune response includes anincrease in antimicrobials such as lysozyme, steroids, egg-white avidin,apoprotein, ovomucoid, ovomucin, ovoflavoprotein, ovoinhibitor, andconalbumin (ovotransferrin). In some embodiments, administration of themicrobial compositions to egg-laying poultry or to eggs of egg-layingpoultry results in either a decrease or an increase in egg-whiteproteins, including lysozyme, steroids, egg-white avidin, apoprotein,ovomucoid, ovomucin, ovoflavoprotein, ovoinhibitor, and conalbumin(ovotransferrin).

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in (1) an increasein hatching success, (2) an increase in the incidence of normal chickmorphology, (3) an increase in the incidence of embryo survival, (4) anincrease in chick growth rate and total body mass, wherein any one ofthe increases is an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% relative to poultry or poultry eggs not havingbeen administered a microbial composition of the present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase inhatching success by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 1%, 6%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% relative to poultry or poultry eggs not having been administered amicrobial composition of the present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase inthe incidence of normal chick morphology by at least 1%, 2%, 3%, 4%, 5%,6%, 7% 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% relative to poultry or poultry eggs nothaving been administered a microbial composition of the presentdisclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase inthe incidence of embryo survival by at least 1%, 2%3, 3%, 4%, 5%, 6%7,7% 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% relative to poultry or poultry eggs nothaving been administered a microbial composition of the presentdisclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase inchick growth rate by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% relative to poultry or poultry eggs not having beenadministered a microbial composition of the present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase inchick total body mass by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% relative to poultry or poultry eggs not having beenadministered a microbial composition of the present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase ordecrease in the concentration of lysozyme present in the egg by at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to poultryor poultry eggs not having been administered a microbial composition ofthe present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase ordecrease in the concentration of steroids present in the egg by at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to poultryor poultry eggs not having been administered a microbial composition ofthe present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase ordecrease in the concentration of avidin present in the egg by at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to poultryor poultry eggs not having been administered a microbial composition ofthe present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase ordecrease in the concentration of apoprotein present in the egg by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7% 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative topoultry or poultry eggs not having been administered a microbialcomposition of the present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase ordecrease in the concentration of ovomucoid present in the egg by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7% 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative topoultry or poultry eggs not having been administered a microbialcomposition of the present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase ordecrease in the concentration of ovomucin present in the egg by at least1%, 2%, 3%, 4%, 5%, 6%, 7% 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to poultryor poultry eggs not having been administered a microbial composition ofthe present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase ordecrease in the concentration of ovoflavoprotein present in the egg byat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relativeto poultry or poultry eggs not having been administered a microbialcomposition of the present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase ordecrease in the concentration of ovoinhibitor present in the egg by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative topoultry or poultry eggs not having been administered a microbialcomposition of the present disclosure.

In some embodiments, the administration of microbial compositions of thepresent disclosure to poultry or poultry eggs results in an increase ordecrease in the concentration of conalbumin present in the egg by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative topoultry or poultry eggs not having been administered a microbialcomposition of the present disclosure.

Encapsulation Compositions

In some embodiments, the microbes or microbial compositions of thedisclosure are encapsulated in an encapsulating composition. Anencapsulating composition protects the microbes from external stressorsprior to entering the gastrointestinal tract of poultry. In someembodiments, external stressors include thermal, desiccating, andphysical stressors associated with pelleting and extrusion. In someembodiments, external stressors include chemicals present in thecompositions to which Encapsulating compositions further create anenvironment that may be beneficial to the microbes, such as minimizingthe oxidative stresses of an aerobic environment on anaerobic microbes,preserving the viability of the microbes wherein vegetative cells orspores form during the pelleting/extrusion process, etc. See Kalsta etal. (U.S. Pat. No. 5,104,662A), Ford (U.S. Pat. No. 5,733,568A), andMosbach and Nilsson (U.S. Pat. No. 4,647,536A) for encapsulationcompositions of microbes, and methods of encapsulating microbes.

In one embodiment, the compositions of the present disclosure exhibit athermal tolerance, which is used interchangeably with heat tolerance andheat resistance. In one embodiment, thermal tolerant compositions of thepresent disclosure are tolerant of the high temperatures associated withfeed manufacturing, mixing of feed and compositions of the presentdisclosure, storage in high heat environments, etc. In one embodiment,thermal tolerant compositions of the present disclosure are resistant toheat-killing and denaturation of the cell wall components and theintracellular environment. In one embodiment, the compositions of thepresent disclosure is tolerant or resistant to dessication/water loss.

In one embodiments, the encapsulation is a reservoir-type encapsulation.In one embodiment, the encapsulation is a matrix-type encapsulation. Inone embodiment, the encapsulation is a coated matrix-type encapsulation.Burgain et al. (2011. J. Food Eng. 104:467-483) discloses numerousencapsulation embodiments and techniques, all of which are incorporatedby reference.

In some embodiments, the compositions of the present disclosure areencapsulated in one or more of the following: gellan gum, xanthan gum,K-Carrageenan, cellulose acetate phthalate, chitosan, starch, milk fat,whey protein, Ca-alginate, raftilose, raftiline, pectin, saccharide,glucose, maltodextrin, gum arabic, guar, seed flour, alginate, dextrins,dextrans, celluloase, gelatin, gelatin, albumin, casein, gluten, acaciagum, tragacanth, wax, paraffin, stearic acid, monodiglycerides, anddiglycerides. In some embodiments, the compositions of the presentdisclosure are encapsulated by one or more of a polymer, carbohydrate,sugar, plastic, glass, polysaccharide, lipid, wax, oil, fatty acid, orglyceride. In one embodiment, the microbial composition is encapsulatedby a glucose. In one embodiment, the microbial composition isencapsulated by a glucose-containing composition. In one embodiment,formulations of the microbial composition comprise a glucoseencapsulant. In one embodiment, formulations of the microbialcomposition comprise a glucose-encapsulated composition.

In some embodiments, the encapsulation of the compositions of thepresent disclosure is carried out by an extrusion, emulsification,coating, agglomeration, lyophilization, vacuum-drying, or spray-drying.

In one embodiment, the encapsulating composition comprises microcapsuleshaving a multiplicity of liquid cores encapsulated in a solid shellmaterial. For purposes of the disclosure, a “multiplicity” of cores isdefined as two or more.

A first category of useful fusible shell materials is that of normallysolid fats, including fats which are already of suitable hardness andanimal or vegetable fats and oils which are hydrogenated until theirmelting points are sufficiently high to serve the purposes of thepresent disclosure. Depending on the desired process and storagetemperatures and the specific material selected, a particular fat can beeither a normally solid or normally liquid material. The terms “normallysolid” and “normally liquid” as used herein refer to the state of amaterial at desired temperatures for storing the resultingmicrocapsules. Since fats and hydrogenated oils do not, strictlyspeaking, have melting points, the term “melting point” is used hereinto describe the minimum temperature at which the fusible materialbecomes sufficiently softened or liquid to be successfully emulsifiedand spray cooled, thus roughly corresponding to the maximum temperatureat which the shell material has sufficient integrity to prevent releaseof the choline cores. “Melting point” is similarly defined herein forother materials which do not have a sharp melting point.

Specific examples of fats and oils useful herein (some of which requirehardening) are as follows: animal oils and fats, such as beef tallow,mutton tallow, lamb tallow, lard or pork fat, fish oil, and sperm oil;vegetable oils, such as canola oil, cottonseed oil, peanut oil, cornoil, olive oil, soybean oil, sunflower oil, safflower oil, coconut oil,palm oil, linseed oil, tung oil, and castor oil; fatty acidmonoglycerides and diglycerides; free fatty acids, such as stearic acid,palmitic acid, and oleic acid; and mixtures thereof. The above listingof oils and fats is not meant to be exhaustive, but only exemplary.

Specific examples of fatty acids include linoleic acid, γ-linoleic acid,dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid,vaccenic acid, nervonic acid, mead acid, erucic acid, gondoic acid,elaidic acid, oleic acid, palitoleic acid, stearidonic acid,eicosapentaenoic acid, valeric acid, caproic acid, enanthic acid,caprylic acid, pelargonic acid, capric acid, undecylic acid, lauricacid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, nonadecyclic acid, arachidic acid,heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid,pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid,nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid,psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid,heptatriacontanoic acid, and octatriacontanoic acid.

Another category of fusible materials useful as encapsulating shellmaterials is that of waxes. Representative waxes contemplated for useherein are as follows: animal waxes, such as beeswax, lanolin, shellwax, and Chinese insect wax; vegetable waxes, such as carnauba,candelilla, bayberry, and sugar cane; mineral waxes, such as paraffin,microcrystalline petroleum, ozocerite, ceresin, and montan; syntheticwaxes, such as low molecular weight polyolefin (e.g., CARBOWAX), andpolyol ether-esters (e.g., sorbitol); Fischer-Tropsch process syntheticwaxes; and mixtures thereof. Water-soluble waxes, such as CARBOWAX andsorbitol, are not contemplated herein if the core is aqueous.

Still other fusible compounds useful herein are fusible natural resins,such as rosin, balsam, shellac, and mixtures thereof.

Various adjunct materials are contemplated for incorporation in fusiblematerials according to the present disclosure. For example,antioxidants, light stabilizers, dyes and lakes, flavors, essentialoils, anti-caking agents, fillers, pH stabilizers, sugars(monosaccharides, disaccharides, trisaccharides, and polysaccharides)and the like can be incorporated in the fusible material in amountswhich do not diminish its utility for the present disclosure.

The core material contemplated herein constitutes from about 0.1% toabout 50%, about 1% to about 35%. or about 5% to about 30% by weight ofthe microcapsules. In some embodiments, the core material contemplatedherein constitutes no more than about 30% by weight of themicrocapsules. In some embodiments, the core material contemplatedherein constitutes about 5% by weight of the microcapsules. The corematerial is contemplated as either a liquid or solid at contemplatedstorage temperatures of the microcapsules.

The cores may include other additives well-known in the pharmaceuticalart, including edible sugars, such as sucrose, glucose, maltose,fructose, lactose, cellobiose, monosaccharides, disaccharides,trisaccharides, and polysaccharides, and mixtures thereof; artificialsweeteners, such as aspartame, saccharin, cyclamate salts, and mixturesthereof; edible acids, such as acetic acid (vinegar), citric acid,ascorbic acid, tartaric acid, and mixtures thereof; edible starches,such as corn starch; hydrolyzed vegetable protein; water-solublevitamins, such as Vitamin C; water-soluble medicaments; water-solublenutritional materials, such as ferrous sulfate; flavors; salts;monosodium glutamate; antimicrobial agents, such as sorbic acid;antimycotic agents, such as potassium sorbate, sorbic acid, sodiumbenzoate, and benzoic acid; food grade pigments and dyes; and mixturesthereof. Other potentially useful supplemental core materials will beapparent to those of ordinary skill in the art.

Emulsifying agents may be employed to assist in the formation of stableemulsions. Representative emulsifying agents include glycerylmonostearate, polysorbate esters, ethoxylated mono- and diglycerides,and mixtures thereof.

For ease of processing, and particularly to enable the successfulformation of a reasonably stable emulsion, the viscosities of the corematerial and the shell material should be similar at the temperature atwhich the emulsion is formed. In particular, the ratio of the viscosityof the shell to the viscosity of the core, expressed in centipoise orcomparable units, and both measured at the temperature of the emulsion,should be from about 22:1 to about 1:1, desirably from about 8:1 toabout 1:1, and preferably from about 3:1 to about 1:1. A ratio of 1:1would be ideal, but a viscosity ratio within the recited ranges isuseful.

Encapsulating compositions are not limited to microcapsule compositionsas disclosed above. In some embodiments encapsulating compositionsencapsulate the microbial compositions in an adhesive polymer that canbe natural or synthetic without toxic effect. In some embodiments, theencapsulating composition may be a matrix selected from sugar matrix,gelatin matrix, polymer matrix, silica matrix, starch matrix, foammatrix, glass/glassy matrix etc. See Pirzio et al. (U.S. Pat. No.7,488,503). In some embodiments, the encapsulating composition may beselected from polyvinyl acetates; polyvinyl acetate copolymers; ethylenevinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcoholcopolymers; celluloses, including ethylcelluloses, methylcelluloses,hydroxymethylcelluloses, hydroxypropylcelluloses andcarboxymethylcellulose; polyvinylpyrolidones; polysaccharides, includingstarch, modified starch, dextrins, maltodextrins, alginate andchitosans; monosaccharides; fats; fatty acids, including oils; proteins,including gelatin and zeins; gum arabics; shellacs; vinylidene chlorideand vinylidene chloride copolymers; calcium lignosulfonates; acryliccopolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymersand copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers;and polychloroprene.

In some embodiments, the microbial composition or a subcomponent thereofis encapsulated in a solid glass matrix or a flexible glass matrix(rubber matrix) comprising one or more polysaccharides, one or moresaccharides, and/or one or more sugar alcohols. In some embodiments, thematrix comprises a monosaccharide or a disaccharide. In someembodiments, the disaccharide may be selected from sucrose, maltose,lactose, lactulose, trehalose, cellobiose, and chitobiose. In someembodiments, the polysaccharides, saccharides, and/or sugar alcohols areadded to the microbial composition or a subcomponent thereofexogenously. In some embodiments, the matrix is an amorphous matrix. Insome embodiments, the microbial composition or a subcomponent thereof isvitrified. In some embodiments, the microbial composition or asubcomponent thereof is desiccated. In some embodiments, the microbialcomposition or a subcomponent thereof is lyophilized. In someembodiments, the microbial composition or a subcomponent thereof isspray dried. In some embodiments, the microbial composition or asubcomponent thereof is spray congealed. In some embodiments, themicrobial composition is preserved/stabilized by preservation byvaporization. See Harel and Kohavi-Beck (U.S. Pat. No. 8,097,245). SeeBronshtein (U.S. Pat. No. 9,469,835).

In some embodiments, the encapsulating compositions comprise at leastone layer of encapsulation. In some embodiments, the encapsulatingcompositions comprise at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 layers ofencapsulation/encapsulants.

In some embodiments, the encapsulating compositions comprise at leasttwo layers of encapsulation. In some embodiments, each layer ofencapsulation confers a different characteristic to the composition. Insome embodiments, no two consecutive layers confer the samecharacteristic. In some embodiments, at least one layer of the at leasttwo layers of encapsulation confers thermostability, shelf stability,ultraviolet resistance, moisture resistance, dessication resistance,hydrophobicity, hydrophilicity, lipophobicity, lipophilicity, pHstability, acid resistance, and base resistance.

In some embodiments, the encapsulating compositions comprise two layersof encapsulation; the first layer confers thermostability and/or shelfstability, and the second layer provides pH resistance.

In some embodiments, the encapsulating layers confer a timed release ofthe microbial composition held in the center of the encapsulatinglayers. In some embodiments, the greater the number of layers confers agreater amount of time before the microbial composition is exposed, postadministration.

In some embodiments, the encapsulating shell of the present disclosurecan be up to 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540μm, 550 μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720μm, 730 μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810μm, 820 μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900μm, 910 μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990μm, 1000 μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070μm, 1080 μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150μm, 1160 μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230μm, 1240 μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310μm, 1320 μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390μm, 1400 μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470μm, 1480 μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm, 1550μm, 1560 μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630μm, 1640 μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710μm, 1720 μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790μm, 1800 μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870μm, 1880 μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950μm, 1960 μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030μm, 2040 μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110μm, 2120 μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190μm, 2200 μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270μm, 2280 μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350μm, 2360 μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm, 2420 μm, 2430μm, 2440 μm, 2450 μm, 2460 μm, 2470 μm, 2480 μm, 2490 μm, 2500 μm, 2510μm, 2520 μm, 2530 μm, 2540 μm, 2550 μm, 2560 μm, 2570 μm, 2580 μm, 2590μm, 2600 μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670μm, 2680 μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750μm, 2760 μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830μm, 2840 μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910μm, 2920 μm, 2930 μm, 2940 μm, 2950 μm, 2960 μm, 2970 μm, 2980 μm, 2990μm, or 3000 μm thick.

Animal Feed

In some embodiments, compositions of the present disclosure are mixedwith animal feed. In some embodiments, animal feed may be present invarious forms such as pellets, capsules, granulated, powdered, mash,liquid, or semi-liquid.

In some embodiments, compositions of the present disclosure are mixedinto the premix or mash at the feed mill, alone as a standalone premix,and/or alongside other feed additives such as MONENSIN, vitamins,antibiotics, etc. In one embodiment, the compositions of the presentdisclosure are mixed into or onto the feed at the feed mill. In anotherembodiment, compositions of the present disclosure are mixed into thefeed itself.

In some embodiments, feed of the present disclosure may be supplementedwith water, premix or premixes, forage, fodder, beans (e.g., whole,cracked, or ground), grains (e.g., whole, cracked, or ground), bean- orgrain-based oils, bean- or grain-based meals, bean- or grain-basedhaylage or silage, bean- or grain-based syrups, fatty acids, sugaralcohols (e.g., polyhydric alcohols), commercially available formulafeeds, oyster shells and those of other bivalves, and mixtures thereof.

In some embodiments, forage encompasses hay, haylage, and silage. Insome embodiments, hays include grass hays (e.g., sudangrass,orchardgrass, or the like), alfalfa hay, and clover hay. In someembodiments, haylages include grass haylages, sorghum haylage, andalfalfa haylage. In some embodiments, silages include maize, oat, wheat,alfalfa, clover, and the like.

In some embodiments, premix or premixes may be utilized in the feed.Premixes may comprise micro-ingredients such as vitamins, minerals,amino acids; chemical preservatives; pharmaceutical compositions such asantibiotics and other medicaments; fermentation products, and otheringredients. In some embodiments, premixes are blended into the feed.

In some embodiments, the feed may include feed concentrates such assoybean hulls, soybean oils, sugar beet pulp, molasses, high proteinsoybean meal, ground corn, shelled corn, wheat midds, distiller grain,cottonseed hulls, and grease. See Anderson et al. (U.S. Pat. No.3,484,243), Iritani et al. (U.S. Pat. No. 6,090,416), Axelrod et al.(U.S. Publication US20060127530A1), and Katsumi et al. (U.S. Pat. No.5,741,508) for animal feed and animal feed supplements capable of use inthe present compositions and methods.

In some embodiments, feed occurs as a compound, which includes, in amixed composition capable of meeting the basic dietary needs, the feeditself, vitamins, minerals, amino acids, and other necessary components.Compound feed may further comprise premixes.

In some embodiments, microbial compositions of the present disclosuremay be mixed with animal feed, premix, and/or compound feed. Individualcomponents of the animal feed may be mixed with the microbialcompositions prior to feeding to poultry. The microbial compositions ofthe present disclosure may be applied into or on a premix, into or on afeed, and/or into or on a compound feed.

Administration of Microbial Compositions

In some embodiments, the microbial compositions of the presentdisclosure are administered to poultry via the oral route. In someembodiments the microbial compositions are administered via a directinjection route into the gastrointestinal tract. In further embodiments,the direct injection administration delivers the microbial compositionsdirectly to one or more of the crop, gizzard, cecum, small intestine,and large intestine. FIG. 3 and FIG. 4 provide a detailed anatomicalview of the gastrointestinal tract of a chicken. In some embodiments,the microbial compositions of the present disclosure are administered toanimals through the cloaca. In further embodiments, cloacaladministration is in the form of an inserted suppository.

In some embodiments, the microbial compositions are administered throughdrinking water, spraying on litter in which the animal is in contactwith, mixing with medications or vaccines, and gavage. In someembodiments, the microbial compositions are sprayed directly on theanimal, wherein the animal ingests the composition having been sprayedon the animal. In some embodiments, the microbial compositions aresprayed directly on the unhatched egg. In some embodiments, themicrobial compositions are sprayed on and/or sprayed in feed, and thefeed is administered to the animal. In further embodiments, the animalingests the composition through the preening of feathers that have comeinto contact with the sprayed composition.

In some embodiments, the microbial compositions are mixed with the feedprior to administration. In some embodiments, the microbial compositionsare pelleted with the feed prior to administration. In some embodiments,the microbial compositions are extruded with the feed prior toadministration. In some aspects, the microbial compositions are mixedinto the feed components as the feed is being prepared. In some aspects,a first group of one or more microbes of the microbial composition arepelleted with the feed, extruded with the feed, and/or mixed into thefeed components as the feed is being prepared. In a further aspect, asecond group of one or more microbes of the microbial composition areadded to the feed which contains the first group of one or moremicrobes.

In some embodiments, the microbial compositions of the presentdisclosure are administered to poultry on day 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, or 31 post-hatching. In some embodiments, the microbialcompositions are administered to the exterior surface of an egg as aliquid, semi-liquid, or solid on day 22, 21, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 pre-hatching. In someembodiments, the microbial compositions of the present disclosure areadministered to poultry in multiple dosing sessions in week(s) 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, and/or 30 week(s) post-hatching. In someembodiments, the microbial compositions are administered immediatelyafter hatching. In some embodiments, the microbial compositions areadministered into the egg (e.g., injection) by itself or administeredalong with other products such as vaccines. In some embodiments, themicrobial compositions of the present disclosure are administered topoultry on hour 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, or 36 post-hatching.

In some embodiments, the microbial compositions are administered one ormore times between the day of hatching and 5 days post-hatching, betweenthe day of hatching and 10 days post-hatching, between the day ofhatching and 14 days post-hatching, between the day of hatching and 16days post-hatching, and between the day of hatching and 24 dayspost-hatching. In a further embodiment, a first microbial composition isadministered one or more times between the day of hatching and 5 dayspost-hatching, between the day of hatching and 10 days post-hatching,between the day of hatching and 14 days post-hatching, between the dayof hatching and 16 days post-hatching, and between the day of hatchingand 24 days post-hatching.

In some embodiments, a first microbial composition is administered topoultry on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31post-hatching. In some embodiments, the first microbial composition isadministered to the exterior surface of an egg as a liquid, semi-liquid,or solid on day 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, 1, or 0 pre-hatching. In some embodiments, thefirst microbial composition of the present disclosure is administered topoultry in multiple dosing sessions in week(s) 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, and/or 30 week(s) post-hatching. In some embodiments, thefirst microbial composition is administered immediately after hatching.In some embodiments, the first microbial composition is administeredinto the egg (e.g., injection) by itself or administered along withother products such as vaccines. In some embodiments, the firstmicrobial composition of the present disclosure is administered topoultry on hour 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, or 36 post-hatching.

In some embodiments, a second or subsequent microbial composition isadministered to poultry on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,or 31 post-hatching. In some embodiments, a second or subsequentmicrobial composition is administered to the exterior surface of an eggas a liquid, semi-liquid, or solid on day 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 pre-hatching. Insome embodiments, a second or subsequent microbial composition of thepresent disclosure is administered to poultry in multiple dosingsessions in week(s) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and/or 30week(s) post-hatching. In some embodiments, a second or subsequentmicrobial composition is administered immediately after hatching. Insome embodiments, a second or subsequent microbial composition isadministered into the egg (e.g., injection) by itself or administeredalong with other products such as vaccines. In some embodiments, asecond or subsequent microbial composition of the present disclosure isadministered to poultry on hour 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, or 36 post-hatching

In some embodiments, the second or subsequent microbial composition isadministered daily for the lifespan of the poultry. In some embodiments,the first microbial composition is administered daily for the lifespanof the poultry. In some embodiments, a microbial composition isadministered daily for the lifespan of the poultry. In some embodiments,the poultry are administered a microbial composition comprising the sametype or types of microbes for the duration of the poultry's life. Infurther embodiments, the microbial composition changes at at least onceover the duration of the poultry's life, but the type or types ofmicrobes in the microbial composition do not change.

In some embodiments, the subsequent microbial composition is a 3^(rd),4^(th), 5^(th), 6^(th), 7^(th), 8^(th), 9^(th), or 10^(th) microbialcomposition, wherein the each of the microbial compositions are distinctfrom one another in the precise strains and/or microbes present in saidcompositions.

In some embodiments, the microbial composition is administered daily forthe lifespan of the poultry. In some embodiments, the microbialcomposition is administered daily for the lifespan of the poultrybeginning on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31post-hatching.

In some embodiments, the microbial composition is administered dailystarting at day 1, 2, 3, 4, 5, 6, or 7 posthatching for the lifespan ofthe poultry. In some embodiments, the microbial composition isadministered daily starting at day 4 or 5 posthatching for the lifespanof the poultry.

In some embodiments, the microbial composition is administered dailystarting at day 1, 2, 3, 4, 5, 6, or 7 posthatching until the first feedchange occurs. In some embodiments, the microbial composition isadministered daily starting at day 4 or 5 until the first feed changeoccurs.

In some embodiments, a first microbial composition is administered dailystarting at day 1, 2, 3, 4, 5, 6, or 7 posthatching until the first feedchange occurs. In some embodiments, a second microbial composition isthen administered daily beginning with the first feed change andspanning the lifespan of the poultry.

In some embodiments, the first microbial composition is administereddaily starting at day 1, 2, 3, 4, 5, 6, or 7 posthatching until thefirst feed change occurs, and the second microbial composition isadministered daily beginning on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 prior to the feed change occurs and continues for the lifespan ofthe poultry. In some embodiments, the first microbial composition isadministered daily starting at day 1, 2, 3, 4, 5, 6, or 7 posthatchinguntil the first feed change occurs, and the second microbial compositionis administered daily beginning on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 after the feed change occurs and continues for the lifespan ofthe poultry.

In some embodiments, the microbial composition is administered daily forthe lifespan of the poultry beginning on week 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 post-hatching. In someembodiments, the microbial composition is administered daily for thelifespan of the poultry beginning 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 week(s) prior to slaughter.

In some embodiments a different microbial composition is administereddaily for the lifespane of the poultry beginning 1, 2, 3, 4, or 5 weeksprior to slaughter, and wherein this microbial composition is differentfrom the first or second microbial compositions administered earlier inthe life of the poultry.

In some embodiments, the first microbial administration is administeredat least once daily until the first feed change occurs. In someembodiments, the first microbial administration is administered at leastonce weekly until the first feed change occurs. In some embodiments, thefirst microbial administration is administered at least twice dailyuntil the first feed change occurs. In some embodiments, the firstmicrobial administration is administered at least once daily for thelife of the poultry. In some embodiments, the first microbialadministration is administered at least once weekly for the life of thepoultry. In some embodiments, the first microbial administration isadministered at least twice daily for the life of the poultry.

In some embodiments, the second microbial administration is administeredat least once daily beginning with the first feed change and spanningthe lifespan of the poultry. In some embodiments, the second microbialadministration is administered at least once weekly beginning with thefirst feed change and spanning the lifespan of the poultry. In someembodiments, the second microbial administration is administered atleast twice daily beginning with the first feed change and spanning thelifespan of the poultry.

In some embodiments, the microbial composition is administered in a dosecomprise a total of, or at least, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml,18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23 ml, 24 ml, 25 ml, 26 ml, 27 ml, 28ml, 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37 ml, 38ml, 39 ml, 40 ml, 41m, 42 ml, 43 ml, 44 ml, 45 ml, 46 ml, 47 ml, 48 ml,49 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, or 1,000 ml.

In some embodiments, the microbial composition is administered in a dosecomprising a total of, or at least, 10¹⁸, 10¹⁷, 10¹⁶, 10¹⁵, 10¹⁴, 10¹³,10¹², 10¹¹, 10¹⁰, 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, or 10² microbialcells.

In some embodiments, the microbial compositions are mixed with feed, andthe administration occurs through the ingestion of the microbialcompositions along with the feed. In some embodiments, the dose of themicrobial composition is administered such that there exists 10² to10¹², 10³ to 10¹², 10⁴ to 10¹², 10⁵ to 10¹², 10⁶ to 10¹², 10⁷ to 10¹²,10⁸ to 10¹², 10⁹ to 10¹², 10¹⁰ to 10¹², 10¹¹ to 10¹², 10² to 10¹¹, 10³to 10¹¹, 10⁴ to 10¹¹, 10⁵ to 10¹¹, 10⁶ to 10¹¹, 10⁷ to 10¹¹, 10⁸ to10¹¹, 10⁹ to 10¹¹, 10¹⁰ to 10¹¹, 10² to 10¹⁰, 10³ to 10¹⁰, 10⁴ to 10¹⁰,10⁵ to 10¹⁰, 10⁶ to 10¹⁰, 10⁷ to 10¹⁰, 10⁸ to 10¹⁰, 10⁹ to 10¹⁰, 10² to10⁹, 10³ to 10⁹, 10⁴ to 10⁹, 10⁵ to 10⁹, 10⁶ to 10⁹, 10⁷ to 10⁹, 10¹⁰ to10⁹, 10² to 10⁸, 10³ to 10⁸, 10⁴ to 10⁸, 10⁵ to 10⁸, 10⁶ to 10⁸, 10⁷ to10⁸, 10² to 10⁷, 10³ to 10⁷, 10⁴ to 10⁷, 10⁵ to 10⁷, 10⁶ to 10⁷, 10² to10⁶, 10³ to 10⁶, 10⁴ to 10⁶, 10⁵ to 10⁶, 10² to 10⁵, 10³ to 10⁵, 10⁴ to10⁵, 10² to 10⁴, 10³ to 10⁴, 10² to 10³, 10¹², 10¹¹, 10¹⁰, 10⁹, 10⁸,10⁷, 10⁶, 10⁵, 10⁴, 10³, or 10² total microbial cells per gram ormilliliter of the composition.

In some embodiments, the administered dose of the microbial compositioncomprises 10² to 10¹⁸, 10³ to 10¹⁸, 10⁴ to 10¹⁸, 10⁵ to 10¹⁸, 10⁶ to10¹⁸, 10⁷ to 10¹⁸, 10⁸ to 10¹⁸, 10⁹ to 10¹⁸, 10¹⁰ to 10¹⁸, 10¹¹ to 10¹⁸,10¹² to 10¹⁸, 10¹³ to 10¹⁸, 10¹⁴ to 10¹⁸, 10¹⁵ to 10¹⁸, 10¹⁶ to 10¹⁸,10¹⁷ to 10¹⁸, 10² to 10¹², 10³ to 10¹², 10⁴ to 10¹², 10⁵ to 10¹², 10⁶ to10¹², 10⁷ to 10¹², 10⁸ to 10¹², 10⁹ to 10¹², 10¹⁰ to 10¹², 10¹¹ to 10¹²,10² to 10¹¹, 10³ to 10¹¹, 10⁴ to 10¹¹, 10⁵ to 10¹¹, 10⁶ to 10¹¹, 10⁷ to10¹¹, 10⁸ to 10¹¹, 10⁹ to 10¹¹, 10¹⁰ to 10¹¹, 10² to 10¹⁰, 10³ to 10¹⁰,10⁴ to 10¹⁰, 10⁵ to 10¹⁰, 10⁶ to 10¹⁰, 10⁷ to 10¹⁰, 10⁸ to 10¹⁰, 10⁹ to10¹⁰, 10² to 10⁹, 10³ to 10⁹, 10⁴ to 10⁹, 10⁵ to 10⁹, 10⁶ to 10⁹, 10⁷ to10⁹, 10⁸ to 10⁹, 10² to 10⁸, 10³ to 10⁸, 10⁴ to 10⁸, 10⁵ to 10⁸, 10⁶ to10⁸, 10⁷ to 10⁸, 10² to 10⁷, 10³ to 10⁷, 10⁴ to 10⁷, 10⁵ to 10⁷, 10⁶ to10⁷, 10² to 10⁶, 10³ to 10⁶, 10⁴ to 10⁶, 10⁵ to 10⁶, 10² to 10⁵, 10³ to10⁵, 10⁴ to 10⁵, 10² to 10⁴, 10³ to 10⁴, 10² to 10³, 10¹⁸, 10¹⁷, 10¹⁶,10¹⁵, 10¹⁴, 10¹³, 10¹², 10¹¹, 10¹⁰, 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³,or 10² total microbial cells.

In some embodiments, the composition is administered 1 or more times perday. In some aspects, the composition is administered with food eachtime the animal is fed. In some embodiments, the composition isadministered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10,4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7,5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per week.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per month.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per year.

In some embodiments, the feed can be uniformly coated with one or morelayers of the microbes and/or microbial compositions disclosed herein,using conventional methods of mixing, spraying, or a combination thereofthrough the use of treatment application equipment that is specificallydesigned and manufactured to accurately, safely, and efficiently applycoatings. Such equipment uses various types of coating technology suchas rotary coaters, drum coaters, fluidized bed techniques, spouted beds,rotary mists, or a combination thereof. Liquid treatments such as thoseof the present disclosure can be applied via either a spinning“atomizer” disk or a spray nozzle, which evenly distributes themicrobial composition onto the feed as it moves though the spraypattern. In some aspects, the feed is then mixed or tumbled for anadditional period of time to achieve additional treatment distributionand drying.

In some embodiments, the feed coats of the present disclosure can be upto 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540 μm, 550μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 μm, 730μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810 μm, 820μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900 μm, 910μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990 μm, 1000μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070 μm, 1080μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150 μm, 1160μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230 μm, 1240μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310 μm, 1320μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390 μm, 1400μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470 μm, 1480μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm, 1550 μm, 1560μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630 μm, 1640μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710 μm, 1720μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790 μm, 1800μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870 μm, 1880μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950 μm, 1960μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030 μm, 2040μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110 μm, 2120μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190 μm, 2200μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270 μm, 2280μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350 μm, 2360μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm, 2420 μm, 2430 μm, 2440μm, 2450 μm, 2460 μm, 2470 μm, 2480 μm, 2490 μm, 2500 μm, 2510 μm, 2520μm, 2530 μm, 2540 μm, 2550 μm, 2560 μm, 2570 μm, 2580 μm, 2590 μm, 2600μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670 μm, 2680μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750 μm, 2760μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830 μm, 2840μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910 μm, 2920μm, 2930 μm, 2940 μm, 2950 μm, 2960 μm, 2970 μm, 2980 μm, 2990 μm, or3000 μm thick.

In some embodiments, the microbial cells can be coated freely onto anynumber of compositions or they can be formulated in a liquid or solidcomposition before being coated onto a composition. For example, a solidcomposition comprising the microorganisms can be prepared by mixing asolid carrier with a suspension of spores or vegetative cells until thesolid carriers are impregnated with the spore or cell suspension. Thismixture can then be dried to obtain the desired particles.

In some other embodiments, it is contemplated that the solid or liquidmicrobial compositions of the present disclosure further containfunctional agents e.g., activated carbon, minerals, vitamins, enzymes,antibiotics and/or other agents capable of improving the quality of theproducts or a combination thereof.

Methods of coating and compositions in use of said methods that areknown in the art can be particularly useful when they are modified bythe addition of one of the embodiments of the present disclosure. Suchcoating methods and apparatus for their application are disclosed in,for example: U.S. Pat. Nos. 8,097,245, 7,998,502, 9,044,497, 8,968,721,9,737,578, 9,469,835, and 5,766,520; and PCT Pat. App. Publication Nos.WO 2008/076975, WO 2010/138522, WO2011/094469, WO 2010/111347, and WO2010/111565, each of which is incorporated by reference herein.

In some embodiments, the microbes or microbial bioensembles of thepresent disclosure exhibit a synergistic effect, on one or more of thetraits described herein, in the presence of one or more of the microbesor bioensembles coming into contact with one another. The synergisticeffect obtained by the taught methods can be quantified, for example,according to Colby's formula (i.e., (E)=X+Y−(X*Y/100)). See Colby, R.S., “Calculating Synergistic and Antagonistic Responses of HerbicideCombinations,” 1967. Weeds. Vol. 15, pp. 20-22, incorporated herein byreference in its entirety. Thus, “synergistic” is intended to reflect anoutcome/parameter/effect that has been increased by more than anadditive amount.

In some embodiments, the microbes or microbial bioensembles of thepresent disclosure may be administered via drench. In one embodiment,the drench is an oral drench. A drench administration comprisesutilizing a drench kit/applicator/syringe that injects/releases a liquidcomprising the microbes or microbial bioensembles into the buccal cavityand/or esophagas of the animal.

In some embodiments, hatchlings/chicks are sprayed with microbialcompositions of the present disclosure between day 0 and day 14 posthatching. In some embodiments, the microbial compositions of the presentdisclosure are administered to the poultry with food. In someembodiments, the spray-administered microbial composition comprises adifferent set of microbes than the microbial composition administeredvia food. In some embodiments, the spray-administered microbialcomposition and the microbial composition administered via food comprisethe same set of microbes.

In some embodiments, the microbes or microbial bioensembles of thepresent disclosure may be administered in a time-released fashion. Thecomposition may be coated in a chemical composition, or may be containedin a mechanical device or capsule that releases the microbes ormicrobial bioensembles over a period of time instead all at once. In oneembodiment, the microbes or microbial bioensembles are administered toan animal in a time-release capsule. In one embodiment, the compositionmay be coated in a chemical composition, or may be contained in amechanical device or capsule that releases the microbes or microbialbioensembles all at once a period of time hours post ingestion. In oneembodiment, the composition may be coated in a chemical composition, ormay be contained in a mechanical device or capsule that releases themicrobes or microbial bioensembles at different locations within thegastrointestinal tract.

In some embodiments, the microbes or microbial bioensembles areadministered in a time-released fashion between 1 to 5, 1 to 10, 1 to15, 1 to 20, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to90, 1 to 95, or 1 to 100 hours post administration of a time-releasecomposition or device.

In some embodiments, the microbes or microbial bioensembles areadministered in a time-released fashion between 1 to 2, 1 to 3, 1 to 4,1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to29, or 1 to 30 days post administration of a time-release composition ordevice.

Microorganisms

As used herein the term “microorganism” should be taken broadly. Itincludes, but is not limited to, the two prokaryotic domains, Bacteriaand Archaea, as well as eukaryotic fungi, protists, and viruses.

By way of example, the microorganisms may include species of the generaof: Lactobacillus, Clostridium, Faecalibacter, Hydrogenoanaerobacterium,Acrocarpospora, Bacillus, Subdoligranulum, Leuconostoc, Lachnospira,Anaerofilum, Microbacterium, Verrucosispora, Blautia, Pseudomonas,Sporobacter, Corynebacterium Streptococcus, Paracoccus, Celulosilyticum,Ruminococcus, Bacteroides, Filobasidium, Gibberella, Alatospora, Pichia,and Candida. In some embodiments, the microorganisms may include speciesof any general disclosed herein.

In certain embodiments, the microorganism is unculturable. This shouldbe taken to mean that the microorganism is not known to be culturable oris difficult to culture using methods known to one skilled in the art.

In one embodiment, the microbes are obtained from animals (e.g.,mammals, reptiles, birds, and the like), soil (e.g., rhizosphere), air,water (e.g., marine, freshwater, wastewater sludge), sediment, oil,plants (e.g., roots, leaves, stems), agricultural products, and extremeenvironments (e.g., acid mine drainage or hydrothermal systems). In afurther embodiment, microbes obtained from marine or freshwaterenvironments such as an ocean, river, or lake. In a further embodiment,the microbes can be from the surface of the body of water, or any depthof the body of water (e.g., a deep sea sample).

The microorganisms of the disclosure may be isolated in substantiallypure or mixed cultures. They may be concentrated, diluted, or providedin the natural concentrations in which they are found in the sourcematerial. For example, microorganisms from saline sediments may beisolated for use in this disclosure by suspending the sediment in freshwater and allowing the sediment to fall to the bottom. The watercontaining the bulk of the microorganisms may be removed by decantationafter a suitable period of settling and either administered to the GItract of poultry, or concentrated by filtering or centrifugation,diluted to an appropriate concentration and administered to the GI tractof poultry with the bulk of the salt removed. By way of further example,microorganisms from mineralized or toxic sources may be similarlytreated to recover the microbes for application to poultry to minimizethe potential for damage to the animal.

In another embodiment, the microorganisms are used in a crude form, inwhich they are not isolated from the source material in which theynaturally reside. For example, the microorganisms are provided incombination with the source material in which they reside; for example,fecal matter or other composition found in the gastrointestinal tract.In this embodiment, the source material may include one or more speciesof microorganisms.

In some embodiments, a mixed population of microorganisms is used in themethods of the disclosure.

In embodiments of the disclosure where the microorganisms are isolatedfrom a source material (for example, the material in which theynaturally reside), any one or a combination of a number of standardtechniques which will be readily known to skilled persons may be used.However, by way of example, these in general employ processes by which asolid or liquid culture of a single microorganism can be obtained in asubstantially pure form, usually by physical separation on the surfaceof a solid microbial growth medium or by volumetric dilutive isolationinto a liquid microbial growth medium. These processes may includeisolation from dry material, liquid suspension, slurries or homogenatesin which the material is spread in a thin layer over an appropriatesolid gel growth medium, or serial dilutions of the material made into asterile medium and inoculated into liquid or solid culture media.

Whilst not essential, in one embodiment, the material containing themicroorganisms may be pre-treated prior to the isolation process inorder to either multiply all microorganisms in the material.Microorganisms can then be isolated from the enriched materials asdisclosed above.

In certain embodiments, as mentioned herein before, the microorganism(s)may be used in crude form and need not be isolated from an animal or amedia. For example, feces, or growth media which includes themicroorganisms identified to be of benefit to increased feed efficiencymay be obtained and used as a crude source of microorganisms for thenext round of the method or as a crude source of microorganisms at theconclusion of the method. For example, fresh feces could be obtained andoptionally processed.

Microbiome Shift and Abundance of Microbes

In some embodiments, the microbiome of poultry, including the gutmicrobiome (crop, gizzard, cecum, small intestine, and large intestine)comprises a diverse environment of microbes with a wide variety ofmetabolic capabilities. The microbiome is influenced by a range offactors including diet, variations in animal metabolism, and breed,among others. Most poultry diets are plant-based and rich in complexpolysaccharides that enrich the gastrointestinal microbial community formicrobes capable of breaking down specific polymeric components in thediet such as cellulose, hemicellulose, lignin, etc. The end products ofprimary degradation sustain a chain of microbes that ultimately producea range of organic acids together with hydrogen and carbon dioxide.Because of the complex and interlinked nature of the microbiome,changing the diet and thus substrates for primary degradation may have acascading effect on gut microbial metabolism, with changes in both theorganic acid profiles and the methane levels produced, thus impactingthe quality and quantity of animal production and/or the productsproduced by the animal. See Menezes et al. (2011. FEMS Microbiol. Ecol.78(2):256-265.)

In some aspects, the present disclosure is drawn to administeringmicrobial compositions described herein to modulate or shift themicrobiome of poultry.

In some embodiments, the microbiome is shifted through theadministration of one or more microbes to the gastrointestinal tract. Infurther embodiments, the one or more microbes are those selected fromTable 1 and/or Table 3. In some embodiments, the microbiome shift ormodulation includes a decrease or loss of specific microbes that werepresent prior to the administration of one or more microbes of thepresent disclosure. In some embodiments, the microbiome shift ormodulation includes an increase in microbes that were present prior tothe administration of one or more microbes of the present disclosure. Insome embodiments, the microbiome shift or modulation includes a gain ofone or more microbes that were not present prior to the administrationof one or more microbes of the present disclosure. In a furtherembodiment, the gain of one or more microbes is a microbe that was notspecifically included in the administered microbial ensemble.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome for a period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 0, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome for a period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome for a period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

In some embodiments, the presence of the administered microbes aredetected by sampling the gastrointestinal tract and using primers toamplify the 16S or 18S rDNA sequences, or the ITS rDNA sequences of theadministered microbes. In some embodiments, the administered microbesare one or more of those selected from Table 1 and/or Table 3, and thecorresponding rDNA sequences are those selected from SEQ ID NOs:1-387.

In some embodiments, the microbiome of a bird is measured by amplifyingpolynucleotides collected from gastrointestinal samples, wherein thepolynucleotides may be 16S or 18S rDNA fragments, or ITS rDNA fragmentsof microbial rDNA. In one embodiment, the microbiome is fingerprinted bya method of denaturing gradient gel electrophoresis (DGGE) wherein theamplified rDNA fragments are sorted by where they denature, and form aunique banding pattern in a gel that may be used for comparing themicrobiome of the same bird over time or the microbiomes of multiplebirds. In another embodiment, the microbiome is fingerprinted by amethod of terminal restriction fragment length polymorphism (T-RFLP),wherein labelled PCR fragments are digested using a restriction enzymeand then sorted by size. In a further embodiment, the data collectedfrom the T-RFLP method is evaluated by nonmetric multidimensionalscaling (nMDS) ordination and PERMANOVA statistics identify differencesin microbiomes, thus allowing for the identification and measurement ofshifts in the microbiome. See also Shanks et al. (2011. Appl. Environ.Microbiol. 77(9):2992-3001), Petri et al. (2013. PLOS one.8(12):e83424), and Menezes et al. (2011. FEMS Microbiol. Ecol.78(2):256-265.)

In some embodiments, the administration of microbes of the presentdisclosure results in a modulation or shift of the microbiome whichfurther results in a desired phenotype or improved trait.

In some embodiments, the decrease in the variability of the number ofunique species is a reduction of the total number of unique species ofmicrobes in the small intestine to between 25 and 500, 25 and 400, 25and 350, 25 and 300, 25 and 200, 25 and 100, 25 and 50, 50 and 500, 50and 400, 50 and 300, 50 and 200, 50 and 100, 100 and 500, 100 and 400,100 and 300, 100 and 200, 200 and 500, 200 and 400, 200 and 300, 300 and500, 300 and 400, or 400 to 500 species.

MIC Scoring

According to the methods provided herein, a sample is processed todetect the presence of one or more microorganism types in the sample(FIG. 1, 1001 ; FIG. 2, 2001 ). The absolute number of one or moremicroorganism organism type in the sample is determined (FIG. 1, 1002 ;

FIG. 2, 2002 ). The determination of the presence of the one or moreorganism types and the absolute number of at least one organism type canbe conducted in parallel or serially. For example, in the case of asample comprising a microbial community comprising bacteria (i.e., onemicroorganism type) and fungi (i.e., a second microorganism type), theuser in one embodiment detects the presence of one or both of theorganism types in the sample (FIG. 1, 1001 ; FIG. 2, 2001 ). The user,in a further embodiment, determines the absolute number of at least oneorganism type in the sample—in the case of this example, the number ofbacteria, fungi or combination thereof, in the sample (FIG. 1, 1002 ;FIG. 2, 2002 ).

In one embodiment, the sample, or a portion thereof is subjected to flowcytometry (FC) analysis to detect the presence and/or number of one ormore microorganism types (FIG. 1, 1001, 1002 ; FIG. 2, 2001, 2002 ). Inone flow cytometer embodiment, individual microbial cells pass throughan illumination zone, at a rate of at least about 300*s⁻¹, or at leastabout 500*s⁻¹, or at least about 1000*s⁻¹. However, one of ordinaryskill in the art will recognize that this rate can vary depending on thetype of instrument is employed. Detectors which are gated electronicallymeasure the magnitude of a pulse representing the extent of lightscattered. The magnitudes of these pulses are sorted electronically into“bins” or “channels,” permitting the display of histograms of the numberof cells possessing a certain quantitative property (e.g., cell stainingproperty, diameter, cell membrane) versus the channel number. Suchanalysis allows for the determination of the number of cells in each“bin” which in embodiments described herein is an “microorganism type”bin, e.g., a bacteria, fungi, nematode, protozoan, archaea, algae,dinoflagellate, virus, viroid, etc.

In one embodiment, a sample is stained with one or more fluorescent dyeswherein a fluorescent dye is specific to a particular microorganismtype, to enable detection via a flow cytometer or some other detectionand quantification method that harnesses fluorescence, such asfluorescence microscopy. The method can provide quantification of thenumber of cells and/or cell volume of a given organism type in a sample.In a further embodiment, as described herein, flow cytometry isharnessed to determine the presence and quantity of a unique firstmarker and/or unique second marker of the organism type, such as enzymeexpression, cell surface protein expression, etc. Two- or three-variablehistograms or contour plots of, for example, light scattering versusfluorescence from a cell membrane stain (versus fluorescence from aprotein stain or DNA stain) may also be generated, and thus animpression may be gained of the distribution of a variety of propertiesof interest among the cells in the population as a whole. A number ofdisplays of such multiparameter flow cytometric data are in common useand are amenable for use with the methods described herein.

In one embodiment of processing the sample to detect the presence andnumber of one or more microorganism types, a microscopy assay isemployed (FIG. 1, 1001, 1002 ). In one embodiment, the microscopy isoptical microscopy, where visible light and a system of lenses are usedto magnify images of small samples. Digital images can be captured by acharge-couple device (CCD) camera. Other microscopic techniques include,but are not limited to, scanning electron microscopy and transmissionelectron microscopy. Microorganism types are visualized and quantifiedaccording to the aspects provided herein.

In another embodiment of in order to detect the presence and number ofone or more microorganism types, the sample, or a portion thereof issubjected to fluorescence microscopy. Different fluorescent dyes can beused to directly stain cells in samples and to quantify total cellcounts using an epifluorescence microscope as well as flow cytometry,described above. Useful dyes to quantify microorganisms include but arenot limited to acridine orange (AO), 4,6-di-amino-2 phenylindole (DAPI)and 5-cyano-2,3 Dytolyl Tetrazolium Chloride (CTC). Viable cells can beestimated by a viability staining method such as the LIVE/DEAD®Bacterial Viability Kit (Bac-Light™) which contains two nucleic acidstains: the green-fluorescent SYTO 9™ dye penetrates all membranes andthe red-fluorescent propidium iodide (PI) dye penetrates cells withdamaged membranes. Therefore, cells with compromised membranes willstain red, whereas cells with undamaged membranes will stain green.Fluorescent in situ hybridization (FISH) extends epifluorescencemicroscopy, allowing for the fast detection and enumeration of specificorganisms. FISH uses fluorescent labelled oligonucleotides probes(usually 15-25 basepairs) which bind specifically to organism DNA in thesample, allowing the visualization of the cells using an epifluorescenceor confocal laser scanning microscope (CLSM). Catalyzed reporterdeposition fluorescence in situ hybridization (CARD-FISH) improves uponthe FISH method by using oligonucleotide probes labelled with a horseradish peroxidase (HRP) to amplify the intensity of the signal obtainedfrom the microorganisms being studied. FISH can be combined with othertechniques to characterize microorganism communities. One combinedtechnique is high affinity peptide nucleic acid (PNA)-FISH, where theprobe has an enhanced capability to penetrate through the ExtracellularPolymeric Substance (EPS) matrix. Another example is LIVE/DEAD-FISHwhich combines the cell viability kit with FISH and has been used toassess the efficiency of disinfection in drinking water distributionsystems.

In another embodiment, the sample, or a portion thereof is subjected toRaman micro-spectroscopy in order to determine the presence of amicroorganism type and the absolute number of at least one microorganismtype (FIG. 1, 1001-1002 ; FIG. 2, 2001-2002 ). Raman micro-spectroscopyis a non-destructive and label-free technology capable of detecting andmeasuring a single cell Raman spectrum (SCRS). A typical SCRS providesan intrinsic biochemical “fingerprint” of a single cell. A SCRS containsrich information of the biomolecules within it, including nucleic acids,proteins, carbohydrates and lipids, which enables characterization ofdifferent cell species, physiological changes and cell phenotypes. Ramanmicroscopy examines the scattering of laser light by the chemical bondsof different cell biomarkers. A SCRS is a sum of the spectra of all thebiomolecules in one single cell, indicating a cell's phenotypic profile.Cellular phenotypes, as a consequence of gene expression, usuallyreflect genotypes. Thus, under identical growth conditions, differentmicroorganism types give distinct SCRS corresponding to differences intheir genotypes and can thus be identified by their Raman spectra.

In yet another embodiment, the sample, or a portion thereof is subjectedto centrifugation in order to determine the presence of a microorganismtype and the number of at least one microorganism type (FIG. 1,1001-1002 ; FIG. 2, 2001-2002 ). This process sediments a heterogeneousmixture by using the centrifugal force created by a centrifuge. Moredense components of the mixture migrate away from the axis of thecentrifuge, while less dense components of the mixture migrate towardsthe axis. Centrifugation can allow fractionation of samples intocytoplasmic, membrane and extracellular portions. It can also be used todetermine localization information for biological molecules of interest.Additionally, centrifugation can be used to fractionate total microbialcommunity DNA. Different prokaryotic groups differ in theirguanine-plus-cytosine (G+C) content of DNA, so density-gradientcentrifugation based on G+C content is a method to differentiateorganism types and the number of cells associated with each type. Thetechnique generates a fractionated profile of the entire community DNAand indicates abundance of DNA as a function of G+C content. The totalcommunity DNA is physically separated into highly purified fractions,each representing a different G+C content that can be analyzed byadditional molecular techniques such as denaturing gradient gelelectrophoresis (DGGE)/amplified ribosomal DNA restriction analysis(ARDRA) (see discussion herein) to assess total microbial communitydiversity and the presence/quantity of one or more microorganism types.

In another embodiment, the sample, or a portion thereof is subjected tostaining in order to determine the presence of a microorganism type andthe number of at least one microorganism type (FIG. 1, 1001-1002 ; FIG.2, 2001-2002 ). Stains and dyes can be used to visualize biologicaltissues, cells or organelles within cells. Staining can be used inconjunction with microscopy, flow cytometry or gel electrophoresis tovisualize or mark cells or biological molecules that are unique todifferent microorganism types. In vivo staining is the process of dyeingliving tissues, whereas in vitro staining involves dyeing cells orstructures that have been removed from their biological context.Examples of specific staining techniques for use with the methodsdescribed herein include, but are not limited to: gram staining todetermine gram status of bacteria, endospore staining to identify thepresence of endospores, Ziehl-Neelsen staining, haematoxylin and eosinstaining to examine thin sections of tissue, papanicolaou staining toexamine cell samples from various bodily secretions, periodicacid-Schiff staining of carbohydrates, Masson's trichome employing athree-color staining protocol to distinguish cells from the surroundingconnective tissue, Romanowsky stains (or common variants that includeWright's stain, Jenner's stain, May-Grunwald stain, Leishman stain andGiemsa stain) to examine blood or bone marrow samples, silver stainingto reveal proteins and DNA, Sudan staining for lipids and Conklin'sstaining to detect true endospores. Common biological stains includeacridine orange for cell cycle determination; bismarck brown for acidmucins; carmine for glycogen; carmine alum for nuclei; Coomassie bluefor proteins; Cresyl violet for the acidic components of the neuronalcytoplasm; Crystal violet for cell walls; DAPI for nuclei; eosin forcytoplasmic material, cell membranes, some extracellular structures andred blood cells; ethidium bromide for DNA; acid fuchsine for collagen,smooth muscle or mitochondria; haematoxylin for nuclei; Hoechst stainsfor DNA; iodine for starch; malachite green for bacteria in the Gimenezstaining technique and for spores; methyl green for chromatin; methyleneblue for animal cells; neutral red for Nissl substance; Nile blue fornuclei; Nile red for lipohilic entities; osmium tetroxide for lipids;rhodamine is used in fluorescence microscopy; safranin for nuclei.Stains are also used in transmission electron microscopy to enhancecontrast and include phosphotungstic acid, osmium tetroxide, rutheniumtetroxide, ammonium molybdate, cadmium iodide, carbohydrazide, ferricchloride, hexamine, indium trichloride, lanthanum nitrate, lead acetate,lead citrate, lead(II) nitrate, periodic acid, phosphomolybdic acid,potassium ferricyanide, potassium ferrocyanide, ruthenium red, silvernitrate, silver proteinate, sodium chloroaurate, thallium nitrate,thiosemicarbazide, uranyl acetate, uranyl nitrate, and vanadyl sulfate.

In another embodiment, the sample, or a portion thereof is subjected tomass spectrometry (MS) in order to determine the presence of amicroorganism type and the number of at least one microorganism type(FIG. 1, 1001-1002 ; FIG. 2, 2001-2002 ). MS, as discussed below, canalso be used to detect the presence and expression of one or more uniquemarkers in a sample (FIG. 1, 1003-1004 ; FIG. 2, 2003-2004 ). MS is usedfor example, to detect the presence and quantity of protein and/orpeptide markers unique to microorganism types and therefore to providean assessment of the number of the respective microorganism type in thesample. Quantification can be either with stable isotope labelling orlabel-free. De novo sequencing of peptides can also occur directly fromMS/MS spectra or sequence tagging (produce a short tag that can bematched against a database). MS can also reveal post-translationalmodifications of proteins and identify metabolites. MS can be used inconjunction with chromatographic and other separation techniques (suchas gas chromatography, liquid chromatography, capillary electrophoresis,ion mobility) to enhance mass resolution and determination.

In another embodiment, the sample, or a portion thereof is subjected tolipid analysis in order to determine the presence of a microorganismtype and the number of at least one microorganism type (FIG. 1,1001-1002 ; FIG. 2, 2001-2002 ). Fatty acids are present in a relativelyconstant proportion of the cell biomass, and signature fatty acids existin microbial cells that can differentiate microorganism types within acommunity. In one embodiment, fatty acids are extracted bysaponification followed by derivatization to give the respective fattyacid methyl esters (FAMEs), which are then analyzed by gaschromatography. The FAME profile in one embodiment is then compared to areference FAME database to identify the fatty acids and theircorresponding microbial signatures by multivariate statistical analyses.

In the aspects of the methods provided herein, the number of uniquefirst makers in the sample, or portion thereof (e.g., sample aliquot) ismeasured, as well as the abundance of each of the unique first markers(FIG. 1, 1003 ; FIG. 2, 2003 ). A unique marker is a marker of amicroorganism strain. It should be understood by one of ordinary skillin the art that depending on the unique marker being probed for andmeasured, the entire sample need not be analyzed. For example, if theunique marker is unique to bacterial strains, then the fungal portion ofthe sample need not be analyzed. As described above, in someembodiments, measuring the absolute abundance of one or more organismtypes in a sample comprises separating the sample by organism type,e.g., via flow cytometry.

Any marker that is unique to an organism strain can be employed herein.For example, markers can include, but are not limited to, small subunitribosomal RNA genes (16S/18S rDNA), large subunit ribosomal RNA genes(23S/25S/28S rDNA), intercalary 5.8S gene, cytochrome c oxidase,beta-tubulin, elongation factor, RNA polymerase and internal transcribedspacer (ITS).

Ribosomal RNA genes (rDNA), especially the small subunit ribosomal RNAgenes, i.e., 18S rRNA genes (18S rDNA) in the case of eukaryotes and 16SrRNA (16S rDNA) in the case of prokaryotes, have been the predominanttarget for the assessment of organism types and strains in a microbialcommunity. However, the large subunit ribosomal RNA genes, 28S rDNAs,have been also targeted. rDNAs are suitable for taxonomic identificationbecause: (i) they are ubiquitous in all known organisms; (ii) theypossess both conserved and variable regions; (iii) there is anexponentially expanding database of their sequences available forcomparison. In community analysis of samples, the conserved regionsserve as annealing sites for the corresponding universal PCR and/orsequencing primers, whereas the variable regions can be used forphylogenetic differentiation. In addition, the high copy number of rDNAin the cells facilitates detection from environmental samples.

The internal transcribed spacer (ITS), located between the 18S rDNA and28S rDNA, has also been targeted. The ITS is transcribed but splicedaway before assembly of the ribosomes The ITS region is composed of twohighly variable spacers, ITS1 and ITS2, and the intercalary 5.85 gene.This rDNA operon occurs in multiple copies in genomes. Because the ITSregion does not code for ribosome components, it is highly variable.

In one embodiment, the unique RNA marker can be an mRNA marker, an siRNAmarker or a ribosomal RNA marker.

Protein-coding functional genes can also be used herein as a uniquefirst marker. Such markers include but are not limited to: therecombinase A gene family (bacterial RecA, archaea RadA and RadB,eukaryotic Rad51 and Rad57, phage UvsX); RNA polymerase R subunit (RpoB)gene, which is responsible for transcription initiation and elongation;chaperonins. Candidate marker genes have also been identified forbacteria plus archaea: ribosomal protein S2 (rpsB), ribosomal proteinS10 (rpsJ), ribosomal protein L1 (rplA), translation elongation factorEF-2, translation initiation factor IF-2, metalloendopeptidase,ribosomal protein L22, ffh signal recognition particle protein,ribosomal protein L4/L1e (rplD), ribosomal protein L2 (rplB), ribosomalprotein S9 (rpsI), ribosomal protein L3 (rplC), phenylalanyl-tRNAsynthetase beta subunit, ribosomal protein L14b/L23e (rplN), ribosomalprotein S5, ribosomal protein S19 (rpsS), ribosomal protein S7,ribosomal protein L16/L10E (rplP), ribosomal protein S13 (rpsM),phenylalanyl-tRNA synthetase a subunit, ribosomal protein L15, ribosomalprotein L25/L23, ribosomal protein L6 (rplF), ribosomal protein L11(rplK), ribosomal protein L5 (rplE), ribosomal protein S12/S23,ribosomal protein L29, ribosomal protein S3 (rpsC), ribosomal proteinS11 (rpsK), ribosomal protein L10, ribosomal protein S8, tRNApseudouridine synthase B, ribosomal protein L18P/L5E, ribosomal proteinS15P/S13e, Porphobilinogen deaminase, ribosomal protein 517, ribosomalprotein L13 (rplM), phosphoribosylformylglycinamidine cyclo-ligase(rpsE), ribonuclease HII and ribosomal protein L24. Other candidatemarker genes for bacteria include: transcription elongation protein NusA(nusA), rpoB DNA-directed RNA polymerase subunit beta (rpoB),GTP-binding protein EngA, rpoC DNA-directed RNA polymerase subunitbeta', priA primosome assembly protein, transcription-repair couplingfactor, CTP synthase (pyrG), secY preprotein translocase subunit SecY,GTP-binding protein Obg/CgtA, DNA polymerase I, rpsF 30S ribosomalprotein S6, poA DNA-directed RNA polymerase subunit alpha, peptide chainrelease factor 1, rplI 50S ribosomal protein L9, polyribonucleotidenucleotidyltransferase, tsf elongation factor Ts (tsf), rplQ 50Sribosomal protein L17, tRNA (guanine-N(1)−)-methyltransferase (rplS),rplY probable 50S ribosomal protein L25, DNA repair protein RadA,glucose-inhibited division protein A, ribosome-binding factor A, DNAmismatch repair protein MutL, smpB SsrA-binding protein (smpB),N-acetylglucosaminyl transferase, S-adenosyl-methyltransferase MraW,UDP-N-acetylmuramoylalanine-D-glutamate ligase, rplS 50S ribosomalprotein L19, rplT 50S ribosomal protein L20 (rplT), ruvA Hollidayjunction DNA helicase, ruvB Holliday junction DNA helicase B, serSseryl-tRNA synthetase, rplU 50S ribosomal protein L21, rpsR 30Sribosomal protein S18, DNA mismatch repair protein MutS, rpsT 30Sribosomal protein S20, DNA repair protein RecN, frr ribosome recyclingfactor (frr), recombination protein RecR, protein of unknown functionUPF0054, miaA tRNA isopentenyltransferase, GTP-binding protein YchF,chromosomal replication initiator protein DnaA, dephospho-CoA kinase,16S rRNA processing protein RimM, ATP-cone domain protein,1-deoxy-D-xylulose 5-phosphate reductoisomerase, 2C-methyl-D-erythritol2,4-cyclodiphosphate synthase, fatty acid/phospholipid synthesis proteinPlsX, tRNA(Ile)-lysidine synthetase, dnaG DNA primase (dnaG), ruvCHolliday junction resolvase, rpsP 30S ribosomal protein S16, RecombinaseA recA, riboflavin biosynthesis protein RibF, glycyl-tRNA synthetasebeta subunit, trmU tRNA(5-methylaminomethyl-2-thiouridylate)-methyltransferase, rpmI 50Sribosomal protein L35, hemE uroporphyrinogen decarboxylase, Rodshape-determining protein, rpmA 50S ribosomal protein L27 (rpmA),peptidyl-tRNA hydrolase, translation initiation factor IF-3 (infC),UDP-N-acetylmuramyl-tripeptide synthetase, rpmF 50S ribosomal proteinL32, rpIL 50S ribosomal protein L7/L12 (rpIL), leuS leucyl-tRNAsynthetase, ligA NAD-dependent DNA ligase, cell division protein FtsA,GTP-binding protein TypA, ATP-dependent Clp protease, ATP-bindingsubunit ClpX, DNA replication and repair protein RecF andUDP-N-acetylenolpyruvoylglucosamine reductase.

Phospholipid fatty acids (PLFAs) may also be used as unique firstmarkers according to the methods described herein. Because PLFAs arerapidly synthesized during microbial growth, are not found in storagemolecules and degrade rapidly during cell death, it provides an accuratecensus of the current living community. All cells contain fatty acids(FAs) that can be extracted and esterified to form fatty acid methylesters (FAMEs). When the FAMEs are analyzed using gaschromatography-mass spectrometry, the resulting profile constitutes a‘fingerprint’ of the microorganisms in the sample. The chemicalcompositions of membranes for organisms in the domains Bacteria andEukarya are comprised of fatty acids linked to the glycerol by anester-type bond (phospholipid fatty acids (PLFAs)). In contrast, themembrane lipids of Archaea are composed of long and branchedhydrocarbons that are joined to glycerol by an ether-type bond(phospholipid ether lipids (PLELs)). This is one of the most widely usednon-genetic criteria to distinguish the three domains. In this context,the phospholipids derived from microbial cell membranes, characterizedby different acyl chains, are excellent signature molecules, becausesuch lipid structural diversity can be linked to specific microbialtaxa.

As provided herein, in order to determine whether an organism strain isactive, the level of expression of one or more unique second markers,which can be the same or different as the first marker, is measured(FIG. 1, 1004 ; FIG. 2, 2004 ). Unique first unique markers aredescribed above. The unique second marker is a marker of microorganismactivity. For example, in one embodiment, the mRNA or protein expressionof any of the first markers described above is considered a uniquesecond marker for the purposes of this invention.

In one embodiment, if the level of expression of the second marker isabove a threshold level (e.g., a control level) or at a threshold level,the microorganism is considered to be active (FIG. 1, 1005 ; FIG. 2,2005 ). Activity is determined in one embodiment, if the level ofexpression of the second marker is altered by at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, or at least about 30%, as compared to a threshold level, which insome embodiments, is a control level.

Second unique markers are measured, in one embodiment, at the protein,RNA or metabolite level. A unique second marker is the same or differentas the first unique marker.

As provided above, a number of unique first markers and unique secondmarkers can be detected according to the methods described herein.Moreover, the detection and quantification of a unique first marker iscarried out according to methods known to those of ordinary skill in theart (FIG. 1, 1003-1004 , FIG. 2, 2003-2004 ).

Nucleic acid sequencing (e.g., gDNA, cDNA, rRNA, mRNA) in one embodimentis used to determine absolute abundance of a unique first marker and/orunique second marker. Sequencing platforms include, but are not limitedto, Sanger sequencing and high-throughput sequencing methods availablefrom Roche/454 Life Sciences, Illumina/Solexa, Pacific Biosciences, IonTorrent and Nanopore. The sequencing can be amplicon sequencing ofparticular DNA or RNA sequences or whole metagenome/transcriptomeshotgun sequencing.

Traditional Sanger sequencing (Sanger et al. (1977) DNA sequencing withchain-terminating inhibitors. Proc Natl. Acad. Sci. USA, 74, pp.5463-5467, incorporated by reference herein in its entirety) relies onthe selective incorporation of chain-terminating dideoxynucleotides byDNA polymerase during in vitro DNA replication and is amenable for usewith the methods described herein.

In another embodiment, the sample, or a portion thereof is subjected toextraction of nucleic acids, amplification of DNA of interest (such asthe rRNA gene) with suitable primers and the construction of clonelibraries using sequencing vectors. Selected clones are then sequencedby Sanger sequencing and the nucleotide sequence of the DNA of interestis retrieved, allowing calculation of the number of unique microorganismstrains in a sample.

454 pyrosequencing from Roche/454 Life Sciences yields long reads andcan be harnessed in the methods described herein (Margulies et al.(2005) Nature, 437, pp. 376-380; U.S. Pat. Nos. 6,274,320; 6,258,568;6,210,891, each of which is herein incorporated in its entirety for allpurposes). Nucleic acid to be sequenced (e.g., amplicons or nebulizedgenomic/metagenomic DNA) have specific adapters affixed on either end byPCR or by ligation. The DNA with adapters is fixed to tiny beads(ideally, one bead will have one DNA fragment) that are suspended in awater-in-oil emulsion. An emulsion PCR step is then performed to makemultiple copies of each DNA fragment, resulting in a set of beads inwhich each bead contains many cloned copies of the same DNA fragment.Each bead is then placed into a well of a fiber-optic chip that alsocontains enzymes necessary for the sequencing-by-synthesis reactions.The addition of bases (such as A, C, G, or T) trigger pyrophosphaterelease, which produces flashes of light that are recorded to infer thesequence of the DNA fragments in each well. About 1 million reads perrun with reads up to 1,000 bases in length can be achieved. Paired-endsequencing can be done, which produces pairs of reads, each of whichbegins at one end of a given DNA fragment. A molecular barcode can becreated and placed between the adapter sequence and the sequence ofinterest in multiplex reactions, allowing each sequence to be assignedto a sample bioinformatically.

Illumina/Solexa sequencing produces average read lengths of about 25basepairs (bp) to about 300 bp (Bennett et al. (2005) Pharmacogenomics,6:373-382; Lange et al. (2014). BMC Genomics 15, p. 63; Fadrosh et al.(2014) Microbiome 2, p. 6; Caporaso et al. (2012) ISME J, 6, p.1621-1624; Bentley et al. (2008) Accurate whole human genome sequencingusing reversible terminator chemistry. Nature, 456:53-59). Thissequencing technology is also sequencing-by-synthesis but employsreversible dye terminators and a flow cell with a field of oligosattached. DNA fragments to be sequenced have specific adapters on eitherend and are washed over a flow cell filled with specificoligonucleotides that hybridize to the ends of the fragments. Eachfragment is then replicated to make a cluster of identical fragments.Reversible dye-terminator nucleotides are then washed over the flow celland given time to attach. The excess nucleotides are washed away, theflow cell is imaged, and the reversible terminators can be removed sothat the process can repeat and nucleotides can continue to be added insubsequent cycles. Paired-end reads that are 300 bases in length eachcan be achieved. An Illumina platform can produce 4 billion fragments ina paired-end fashion with 125 bases for each read in a single run.Barcodes can also be used for sample multiplexing, but indexing primersare used.

The SOLiD (Sequencing by Oligonucleotide Ligation and Detection, LifeTechnologies) process is a “sequencing-by-ligation” approach, and can beused with the methods described herein for detecting the presence andabundance of a first marker and/or a second marker (FIG. 1, 1003-1004 ;FIG. 2,2003-2004 ) (Peckham et al. SOLiD™ Sequencing and 2-BaseEncoding. San Diego, CA: American Society of Human Genetics, 2007; Mitraet al. (2013) Analysis of the intestinal microbiota using SOLiD 16S rRNAgene sequencing and SOLiD shotgun sequencing. BMC Genomics, 14(Suppl 5):S16; Mardis (2008) Next-generation DNA sequencing methods. Annu RevGenomics Hum Genet, 9:387-402; each incorporated by reference herein inits entirety). A library of DNA fragments is prepared from the sample tobe sequenced, and are used to prepare clonal bead populations, whereonly one species of fragment will be present on the surface of eachmagnetic bead. The fragments attached to the magnetic beads will have auniversal P1 adapter sequence so that the starting sequence of everyfragment is both known and identical. Primers hybridize to the P1adapter sequence within the library template. A set of fourfluorescently labelled di-base probes compete for ligation to thesequencing primer. Specificity of the di-base probe is achieved byinterrogating every 1st and 2nd base in each ligation reaction. Multiplecycles of ligation, detection and cleavage are performed with the numberof cycles determining the eventual read length. The SOLiD platform canproduce up to 3 billion reads per run with reads that are 75 bases long.Paired-end sequencing is available and can be used herein, but with thesecond read in the pair being only 35 bases long. Multiplexing ofsamples is possible through a system akin to the one used by Illumina,with a separate indexing run.

The Ion Torrent system, like 454 sequencing, is amenable for use withthe methods described herein for detecting the presence and abundance ofa first marker and/or a second marker (FIG. 1, 1003-1004 ; FIG. 2,2003-2004 ). It uses a plate of microwells containing beads to which DNAfragments are attached. It differs from all of the other systems,however, in the manner in which base incorporation is detected. When abase is added to a growing DNA strand, a proton is released, whichslightly alters the surrounding pH. Microdetectors sensitive to pH areassociated with the wells on the plate, and they record when thesechanges occur. The different bases (A, C, G, T) are washed sequentiallythrough the wells, allowing the sequence from each well to be inferred.The Ion Proton platform can produce up to 50 million reads per run thathave read lengths of 200 bases. The Personal Genome Machine platform haslonger reads at 400 bases. Bidirectional sequencing is available.Multiplexing is possible through the standard in-line molecular barcodesequencing.

Pacific Biosciences (PacBio) SMRT sequencing uses a single-molecule,real-time sequencing approach and in one embodiment, is used with themethods described herein for detecting the presence and abundance of afirst marker and/or a second marker (FIG. 1, 1003-1004 ; FIG. 2,2003-2004 ). The PacBio sequencing system involves no amplificationstep, setting it apart from the other major next-generation sequencingsystems. In one embodiment, the sequencing is performed on a chipcontaining many zero-mode waveguide (ZMW) detectors. DNA polymerases areattached to the ZMW detectors and phospholinked dye-labeled nucleotideincorporation is imaged in real time as DNA strands are synthesized. ThePacBio system yields very long read lengths (averaging around 4,600bases) and a very high number of reads per run (about 47,000). Thetypical “paired-end” approach is not used with PacBio, since reads aretypically long enough that fragments, through CCS, can be coveredmultiple times without having to sequence from each end independently.Multiplexing with PacBio does not involve an independent read, butrather follows the standard “in-line” barcoding model.

In one embodiment, where the first unique marker is the ITS genomicregion, automated ribosomal intergenic spacer analysis (ARISA) is usedin one embodiment to determine the number and identity of microorganismstrains in a sample (FIG. 1, 1003 , FIG. 2, 2003 ) (Ranjard et al.(2003). Environmental Microbiology 5, pp. 1111-1120, incorporated byreference in its entirety for all purposes). The ITS region hassignificant heterogeneity in both length and nucleotide sequence. Theuse of a fluorescence-labeled forward primer and an automatic DNAsequencer permits high resolution of separation and high throughput. Theinclusion of an internal standard in each sample provides accuracy insizing general fragments.

In another embodiment, fragment length polymorphism (RFLP) ofPCR-amplified rDNA fragments, otherwise known as amplified ribosomal DNArestriction analysis (ARDRA), is used to characterize unique firstmarkers and the abundance of the same in samples (FIG. 1, 1003 , FIG. 2,2003 ) (Massol-Deya et al. (1995). Mol. Microb. Ecol. Manual. 3.3.2, pp.1-18, incorporated by reference in its entirety for all purposes). rDNAfragments are generated by PCR using general primers, digested withrestriction enzymes, electrophoresed in agarose or acrylamide gels, andstained with ethidium bromide or silver nitrate.

One fingerprinting technique used in detecting the presence andabundance of a unique first marker is single-stranded-conformationpolymorphism (SSCP) (Lee et al. (1996). Appl Environ Microbiol 62, pp.3112-3120; Scheinert et al. (1996). J. Microbiol. Methods 26, pp.103-117; Schwieger and Tebbe (1998). Appl. Environ. Microbiol. 64, pp.4870-4876, each of which is incorporated by reference herein in itsentirety). In this technique, DNA fragments such as PCR productsobtained with primers specific for the 16S rRNA gene, are denatured anddirectly electrophoresed on a non-denaturing gel. Separation is based ondifferences in size and in the folded conformation of single-strandedDNA, which influences the electrophoretic mobility. Reannealing of DNAstrands during electrophoresis can be prevented by a number ofstrategies, including the use of one phosphorylated primer in the PCRfollowed by specific digestion of the phosphorylated strands with lambdaexonuclease and the use of one biotinylated primer to perform magneticseparation of one single strand after denaturation. To assess theidentity of the predominant populations in a given bioensemble, in oneembodiment, bands are excised and sequenced, or SSCP-patterns can behybridized with specific probes. Electrophoretic conditions, such as gelmatrix, temperature, and addition of glycerol to the gel, can influencethe separation.

In addition to sequencing based methods, other methods for quantifyingexpression (e.g., gene, protein expression) of a second marker areamenable for use with the methods provided herein for determining thelevel of expression of one or more second markers (FIG. 1, 1004 ; FIG.2, 2004 ). For example, quantitative RT-PCR, microarray analysis, linearamplification techniques such as nucleic acid sequence basedamplification (NASBA) are all amenable for use with the methodsdescribed herein, and can be carried out according to methods known tothose of ordinary skill in the art.

In another embodiment, the sample, or a portion thereof is subjected toa quantitative polymerase chain reaction (PCR) for detecting thepresence and abundance of a first marker and/or a second marker (FIG. 1,1003-1004 ; FIG. 2, 2003-2004 ). Specific microorganism strains activityis measured by reverse transcription of transcribed ribosomal and/ormessenger RNA (rRNA and mRNA) into complementary DNA (cDNA), followed byPCR (RT-PCR).

In another embodiment, the sample, or a portion thereof is subjected toPCR-based fingerprinting techniques to detect the presence and abundanceof a first marker and/or a second marker (FIG. 1, 1003-1004 ; FIG. 2,2003-2004 ). PCR products can be separated by electrophoresis based onthe nucleotide composition. Sequence variation among the different DNAmolecules influences the melting behaviour, and therefore molecules withdifferent sequences will stop migrating at different positions in thegel. Thus electrophoretic profiles can be defined by the position andthe relative intensity of different bands or peaks and can be translatedto numerical data for calculation of diversity indices. Bands can alsobe excised from the gel and subsequently sequenced to reveal thephylogenetic affiliation of the community members. Electrophoresismethods include, but are not limited to: denaturing gradient gelelectrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE),single-stranded-conformation polymorphism (SSCP), restriction fragmentlength polymorphism analysis (RFLP) or amplified ribosomal DNArestriction analysis (ARDRA), terminal restriction fragment lengthpolymorphism analysis (T-RFLP), automated ribosomal intergenic spaceranalysis (ARISA), randomly amplified polymorphic DNA (RAPD), DNAamplification fingerprinting (DAF) and Bb-PEG electrophoresis.

In another embodiment, the sample, or a portion thereof is subjected toa chip-based platform such as microarray or microfluidics to determinethe abundance of a unique first marker and/or presence/abundance of aunique second marker (FIG. 1, 1003-1004 , FIG. 2, 2003-2004 ). The PCRproducts are amplified from total DNA in the sample and directlyhybridized to known molecular probes affixed to microarrays. After thefluorescently labeled PCR amplicons are hybridized to the probes,positive signals are scored by the use of confocal laser scanningmicroscopy. The microarray technique allows samples to be rapidlyevaluated with replication, which is a significant advantage inmicrobial community analyses. In general, the hybridization signalintensity on microarrays is directly proportional to the abundance ofthe target organism. The universal high-density 16S microarray(PhyloChip) contains about 30,000 probes of 16SrRNA gene targeted toseveral cultured microbial species and “candidate divisions”. Theseprobes target all 121 demarcated prokaryotic orders and allowsimultaneous detection of 8,741 bacterial and archaeal taxa. Anothermicroarray in use for profiling microbial communities is the FunctionalGene Array (FGA). Unlike PhyloChips, FGAs are designed primarily todetect specific metabolic groups of bacteria. Thus, FGA not only revealthe community structure, but they also shed light on the in situcommunity metabolic potential. FGA contain probes from genes with knownbiological functions, so they are useful in linking microbial communitycomposition to ecosystem functions. An FGA termed GeoChipcontains >24,000 probes from all known metabolic genes involved invarious biogeochemical, ecological, and environmental processes such asammonia oxidation, methane oxidation, and nitrogen fixation.

A protein expression assay, in one embodiment, is used with the methodsdescribed herein for determining the level of expression of one or moresecond markers (FIG. 1, 1004 ; FIG. 2, 2004 ). For example, in oneembodiment, mass spectrometry or an immunoassay such as an enzyme-linkedimmunosorbant assay (ELISA) is utilized to quantify the level ofexpression of one or more unique second markers, wherein the one or moreunique second markers is a protein.

In one embodiment, the sample, or a portion thereof is subjected toBromodeoxyuridine (BrdU) incorporation to determine the level of asecond unique marker (FIG. 1, 1004 ; FIG. 2, 2004 ). BrdU, a syntheticnucleoside analog of thymidine, can be incorporated into newlysynthesized DNA of replicating cells. Antibodies specific for BRdU canthen be used for detection of the base analog. Thus BrdU incorporationidentifies cells that are actively replicating their DNA, a measure ofactivity of a microorganism according to one embodiment of the methodsdescribed herein. BrdU incorporation can be used in combination withFISH to provide the identity and activity of targeted cells.

In one embodiment, the sample, or a portion thereof is subjected tomicroautoradiography (MAR) combined with FISH to determine the level ofa second unique marker (FIG. 1, 1004 ;

FIG. 2, 2004 ). MAR-FISH is based on the incorporation of radioactivesubstrate into cells, detection of the active cells usingautoradiography and identification of the cells using FISH. Thedetection and identification of active cells at single-cell resolutionis performed with a microscope. MAR-FISH provides information on totalcells, probe targeted cells and the percentage of cells that incorporatea given radiolabelled substance. The method provides an assessment ofthe in situ function of targeted microorganisms and is an effectiveapproach to study the in vivo physiology of microorganisms. A techniquedeveloped for quantification of cell-specific substrate uptake incombination with MAR-FISH is known as quantitative MAR (QMAR).

In one embodiment, the sample, or a portion thereof is subjected tostable isotope Raman spectroscopy combined with FISH (Raman-FISH) todetermine the level of a second unique marker (FIG. 1, 1004 ; FIG. 2,2004 ). This technique combines stable isotope probing, Ramanspectroscopy and FISH to link metabolic processes with particularorganisms. The proportion of stable isotope incorporation by cellsaffects the light scatter, resulting in measurable peak shifts forlabelled cellular components, including protein and mRNA components.Raman spectroscopy can be used to identify whether a cell synthesizescompounds including, but not limited to: oil (such as alkanes), lipids(such as triacylglycerols (TAG)), specific proteins (such as hemeproteins, metalloproteins), cytochrome (such as P450, cytochrome c),chlorophyll, chromophores (such as pigments for light harvestingcarotenoids and rhodopsins), organic polymers (such aspolyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB)), hopanoids,steroids, starch, sulfide, sulfate and secondary metabolites (such asvitamin B12).

In one embodiment, the sample, or a portion thereof is subjected toDNA/RNA stable isotope probing (SIP) to determine the level of a secondunique marker (FIG. 1, 1004 ; FIG. 2, 2004 ). SIP enables determinationof the microbial diversity associated with specific metabolic pathwaysand has been generally applied to study microorganisms involved in theutilization of carbon and nitrogen compounds. The substrate of interestis labelled with stable isotopes (such as ¹³C or ¹⁵N) and added to thesample. Only microorganisms able to metabolize the substrate willincorporate it into their cells. Subsequently, ¹³C-DNA and ¹⁵N-DNA canbe isolated by density gradient centrifugation and used for metagenomicanalysis. RNA-based SIP can be a responsive biomarker for use in SIPstudies, since RNA itself is a reflection of cellular activity.

In one embodiment, the sample, or a portion thereof is subjected toisotope array to determine the level of a second unique marker (FIG. 1,1004 ; FIG. 2, 2004 ). Isotope arrays allow for functional andphylogenetic screening of active microbial communities in ahigh-throughput fashion. The technique uses a combination of SIP formonitoring the substrate uptake profiles and microarray technology fordetermining the taxonomic identities of active microbial communities.Samples are incubated with a ¹⁴C-labeled substrate, which during thecourse of growth becomes incorporated into microbial biomass. The¹⁴C-labeled rRNA is separated from unlabeled rRNA and then labeled withfluorochromes. Fluorescent labeled rRNA is hybridized to a phylogeneticmicroarray followed by scanning for radioactive and fluorescent signals.The technique thus allows simultaneous study of microbial communitycomposition and specific substrate consumption by metabolically activemicroorganisms of complex microbial communities.

In one embodiment, the sample, or a portion thereof is subjected to ametabolomics assay to determine the level of a second unique marker(FIG. 1, 1004 ; FIG. 2, 2004 ). Metabolomics studies the metabolomewhich represents the collection of all metabolites, the end products ofcellular processes, in a biological cell, tissue, organ or organism.This methodology can be used to monitor the presence of microorganismsand/or microbial mediated processes since it allows associating specificmetabolite profiles with different microorganisms. Profiles ofintracellular and extracellular metabolites associated with microbialactivity can be obtained using techniques such as gaschromatography-mass spectrometry (GC-MS). The complex mixture of ametabolomic sample can be separated by such techniques as gaschromatography, high performance liquid chromatography and capillaryelectrophoresis. Detection of metabolites can be by mass spectrometry,nuclear magnetic resonance (NMR) spectroscopy, ion-mobilityspectrometry, electrochemical detection (coupled to HPLC) and radiolabel(when combined with thin-layer chromatography).

According to the embodiments described herein, the presence andrespective number of one or more active microorganism strains in asample are determined (FIG. 1, 1006 ; FIG. 2, 2006 ). For example,strain identity information obtained from assaying the number andpresence of first markers is analyzed to determine how many occurrencesof a unique first marker are present, thereby representing a uniquemicroorganism strain (e.g., by counting the number of sequence reads ina sequencing assay). This value can be represented in one embodiment asa percentage of total sequence reads of the first maker to give apercentage of unique microorganism strains of a particular microorganismtype. In a further embodiment, this percentage is multiplied by thenumber of microorganism types (obtained at step 1002 or 2002, see FIG. 1and FIG. 2 ) to give the absolute abundance of the one or moremicroorganism strains in a sample and a given volume.

The one or more microorganism strains are considered active, asdescribed above, if the level of second unique marker expression at athreshold level, higher than a threshold value, e.g., higher than atleast about 5%, at least about 10%, at least about 20% or at least about30% over a control level.

In another aspect of the invention, a method for determining theabsolute abundance of one or more microorganism strains is determined ina plurality of samples (FIG. 2 , see in particular, 2007). For amicroorganism strain to be classified as active, it need only be activein one of the samples. The samples can be taken over multiple timepoints from the same source, or can be from different environmentalsources (e.g., different animals).

The absolute abundance values over samples are used in one embodiment torelate the one or more active microorganism strains, with anenvironmental parameter (FIG. 2, 2008 ). In one embodiment, theenvironmental parameter is the presence of a second active microorganismstrain. Relating the one or more active microorganism strains to theenvironmental parameter, in one embodiment, is carried out bydetermining the co-occurrence of the strain and parameter by correlationor by network analysis.

In one embodiment, determining the co-occurrence of one or more activemicroorganism strains with an environmental parameter comprises anetwork and/or cluster analysis method to measure connectivity ofstrains or a strain with an environmental parameter within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In another embodiment, thenetwork and/or cluster analysis method may be applied to determining theco-occurrence of two or more active microorganism strains in a sample(FIG. 2, 2008 ). In another embodiment, the network analysis comprisesnonparametric approaches including mutual information to establishconnectivity between variables. In another embodiment, the networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof (FIG. 2, 2009 ).In another embodiment, the cluster analysis method comprises building aconnectivity model, subspace model, distribution model, density model,or a centroid model and/or using community detection algorithms such asthe Louvain, Bron-Kerbosch, Girvan-Newman, Clauset-Newman-Moore,Pons-Latapy, and Wakita-Tsurumi algorithms (FIG. 2, 2010 ).

In one embodiment, the cluster analysis method is a heuristic methodbased on modularity optimization. In a further embodiment, the clusteranalysis method is the Louvain method. See, e.g., the method describedby Blondel et al. (2008). Fast unfolding of communities in largenetworks. Journal of Statistical Mechanics: Theory and Experiment,Volume 2008, October 2008, incorporated by reference herein in itsentirety for all purposes.

In another embodiment, the network analysis comprises predictivemodeling of network through link mining and prediction, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, the network analysiscomprises differential equation based modeling of populations. Inanother embodiment, the network analysis comprises Lotka-Volterramodeling.

In one embodiment, relating the one or more active microorganism strainsto an environmental parameter (e.g., determining the co-occurrence) inthe sample comprises creating matrices populated with linkages denotingenvironmental parameter and microorganism strain associations.

In one embodiment, the multiple sample data obtained at step 2007 (e.g.,over two or more samples which can be collected at two or more timepoints where each time point corresponds to an individual sample), iscompiled. In a further embodiment, the number of cells of each of theone or more microorganism strains in each sample is stored in anassociation matrix (which can be in some embodiments, an abundancematrix). In one embodiment, the association matrix is used to identifyassociations between active microorganism strains in a specific timepoint sample using rule mining approaches weighted with association(e.g., abundance) data. Filters are applied in one embodiment to removeinsignificant rules.

In one embodiment, the absolute abundance of one or more, or two or moreactive microorganism strains is related to one or more environmentalparameters (FIG. 2, 2008 ), e.g., via co-occurrence determination.Environmental parameters are chosen by the user depending on thesample(s) to be analyzed and are not restricted by the methods describedherein. The environmental parameter can be a parameter of the sampleitself, e.g., pH, temperature, amount of protein in the sample.Alternatively, the environmental parameter is a parameter that affects achange in the identity of a microbial community (i.e., where the“identity” of a microbial community is characterized by the type ofmicroorganism strains and/or number of particular microorganism strainsin a community), or is affected by a change in the identity of amicrobial community. For example, an environmental parameter in oneembodiment, is the food intake of an animal or the amount of eggsproduced by poultry. In one embodiment, the environmental parameter isthe presence, activity and/or abundance of a second microorganism strainin the microbial community, present in the same sample.

In some embodiments described herein, an environmental parameter isreferred to as a metadata parameter.

Other examples of metadata parameters include but are not limited togenetic information from the host from which the sample was obtained(e.g., DNA mutation information), sample pH, sample temperature,expression of a particular protein or mRNA, nutrient conditions (e.g.,level and/or identity of one or more nutrients) of the surroundingenvironment/ecosystem), susceptibility or resistance to disease, onsetor progression of disease, susceptibility or resistance of the sample totoxins, efficacy of xenobiotic compounds (pharmaceutical drugs),biosynthesis of natural products, or a combination thereof.

For example, according to one embodiment, microorganism strain numberchanges are calculated over multiple samples according to the method ofFIG. 2 (i.e., at 2001-2007). Strain number changes of one or more activestrains over time is compiled (e.g., one or more strains that haveinitially been identified as active according to step 2006), and thedirectionality of change is noted (i.e., negative values denotingdecreases, positive values denoting increases). The number of cells overtime is represented as a network, with microorganism strainsrepresenting nodes and the abundance weighted rules representing edges.Markov chains and random walks are leveraged to determine connectivitybetween nodes and to define clusters. Clusters in one embodiment arefiltered using metadata in order to identify clusters associated withdesirable metadata (FIG. 2, 2008 ).

In a further embodiment, microorganism strains are ranked according toimportance by integrating cell number changes over time and strainspresent in target clusters, with the highest changes in cell numberranking the highest.

Network and/or cluster analysis method in one embodiment, is used tomeasure connectivity of the one or more strains within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In one embodiment, networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof. In anotherembodiment, network analysis comprises predictive modeling of networkthrough link mining and prediction, social network theory, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, network analysis comprisesdifferential equation based modeling of populations. In yet anotherembodiment, network analysis comprises Lotka-Volterra modeling.

Cluster analysis method comprises building a connectivity model,subspace model, distribution model, density model, or a centroid model.

Network and cluster based analysis, for example, to carry out methodstep 2008 of FIG. 2 , can be carried out via a module. As used herein, amodule can be, for example, any assembly, instructions and/or set ofoperatively-coupled electrical components, and can include, for example,a memory, a processor, electrical traces, optical connectors, software(executing in hardware) and/or the like.

Network Analysis

A network and/or cluster analysis method, in one embodiment, is used tomeasure connectivity of the one or more strains within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In one embodiment, networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof. In anotherembodiment, network analysis comprises predictive modeling of networkthrough link mining and prediction, social network theory, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, network analysis comprisesmutual information, maximal information coefficient (MIC) calculations,or other nonparametric methods between variables to establishconnectivity. In another embodiment, network analysis comprisesdifferential equation based modeling of populations. In yet anotherembodiment, network analysis comprises Lotka-Volterra modeling.

The environmental parameter can be a parameter of the sample itself,e.g., pH, temperature, amount of protein in the sample. Alternatively,the environmental parameter is a parameter that affects a change in theidentity of a microbial community (i.e., where the “identity” of amicrobial community is characterized by the type of microorganismstrains and/or number of particular microorganism strains in acommunity), or is affected by a change in the identity of a microbialcommunity. For example, an environmental parameter in one embodiment, isthe food intake of an animal or the amount of eggs produced. In oneembodiment, the environmental parameter is the presence, activity and/orabundance of a second microorganism strain in the microbial community,present in the same sample. In some embodiments, an environmentalparameter is referred to as a metadata parameter.

Other examples of metadata parameters include but are not limited togenetic information from the host from which the sample was obtained(e.g., DNA mutation information), sample pH, sample temperature,expression of a particular protein or mRNA, nutrient conditions (e.g.,level and/or identity of one or more nutrients) of the surroundingenvironment/ecosystem), susceptibility or resistance to disease, onsetor progression of disease, susceptibility or resistance of the sample totoxins, efficacy of xenobiotic compounds (pharmaceutical drugs),biosynthesis of natural products, or a combination thereof.

Poultry Pathogen Resistance and Clearance

In some aspects, the present disclosure is drawn to administering one ormore microbial compositions described herein to poultry to clear thegastrointestinal tract of pathogenic microbes. In some embodiments, thepresent disclosure is further drawn to administering microbialcompositions described herein to prevent colonization of pathogenicmicrobes in the gastrointestinal tract. In some embodiments, theadministration of microbial compositions described herein further clearpathogens from the integument and the respiratory tract of poultry,and/or prevent colonization of pathogens on the integument and in therespiratory tract. In some embodiments, the administration of microbialcompositions described herein reduce leaky gut/intestinal permeability,inflammation, and/or incidence of liver disease. In some embodiments,the administration of microbial compositions described herein promotethe development of the immune system.

In some embodiments, the microbial compositions of the presentdisclosure comprise one or more microbes that are present in thegastrointestinal tract of poultry at a relative abundance of less than15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, or 0.01%.

In some embodiments, after administration of microbial compositions ofthe present disclosure the one or more microbes are present in thegastrointestinal tract of the poultry at a relative abundance of atleast 0.5%, 1%, 5%, 10%, 15%, 20%2, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Pathogenic microbes of poultry include the following: Mycoplasmagallisepticum, Mycoplasma meleagridis, Mycoplasma synoviae, Pasteurellamultocida, Clostridium perfringens, Clostridium colinum, Clostridiumbotulinum, Salmonella typi, Salmonella typhimurium, Salmonella enterica,Salmonella pullorum, Salmonella gallinarum, Hemophilus gallinarum,Erysipelothrix insidiosa, Campylobacter jejuni, Campylobacter coli,Campylobacter lari, Listeria monocytogenes, Arcobacter butzleri,Mycobacterium avium, and pathogenic strains of Escherichia coli andStaphylococcus aureus. In some embodiments, the pathogenic microbesinclude viral pathogens. In some embodiments, the pathogenic microbesare pathogenic to both poultry and humans. In some embodiments, thepathogenic microbes are pathogenic to either poultry or humans.

In some embodiments, the administration of compositions of the presentdisclosure to poultry modulate the makeup of the gastrointestinalmicrobiome such that the administered microbes outcompete microbialpathogens present in the gastrointestinal tract. In some embodiments,the administration of compositions of the present disclosure to poultryharboring microbial pathogens outcompetes the pathogens and clears thepoultry of the pathogens. In some embodiments, the administration ofcompositions of the present disclosure stimulate host immunity, and aidsin clearance of the microbial pathogens. In some embodiments, theadministration of compositions of the present disclosure introducemicrobes that produce bacteriostatic and/or bactericidal components thatdecrease or clear the poultry of the microbial pathogens. In someembodiments, the administration of compositions of the presentdisclosure introduces microbes that modulate the pH, nutrientavailability, mineral composition, and/or vitamin composition of thegastrointestinal tract. In some embodiments, the administration ofcompostions of the present disclosure introduces microbes that increasethe gastrointestinal pH, resulting in the inhibition of pathogen growth.In some embodiments, the administration of compostions of the presentdisclosure introduces microbes that decrease the gastrointestinal pH,resulting in the inhibition of pathogen growth.

In some embodiments, challenging poultry with a microbial colonizer ormicrobial pathogen after administering one or more compositions of thepresent disclosure prevents the microbial colonizer or microbialpathogen from growing to a relative abundance of greater than 15%, 4%1,3%1, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or0.01%. In further embodiments, challenging poultry with a microbialcolonizer or microbial pathogen after administering one or morecompositions of the present disclosure prevents the microbial colonizeror microbial pathogen from colonizing poultry

In some embodiments, clearance of the microbial colonizer or microbialpathogen occurs occurs in less than 25 days, less than 24 days, lessthan 23 days, less than 22 days, less than 21 days, less than 20 days,less than 19 days, less than 18 days, less than 17 days, less than 16days, less than 15 days, less than 14 days, less than 13 days, less than12 days, less than 11 days, less than 10 days, less than 9 days, lessthan 8 days, less than 7 days, less than 6 days, less than 5 days, lessthan 4 days, less than 3 days, or less than 2 days post administrationof the one or more compositions of the present disclosure.

In some embodiments, clearance of the microbial colonizer or microbialpathogen occurs within 1-30 days, 1-25 days, 1-20 day, 1-15 days, 1-10days, 1-5 days, 5-30 days, 5-25 days, 5-20 days, 5-15 days, 5-10 days,10-30 days, 10-25 days, 10-20 days, 10-15 days, 15-30 days, 15-25 days,15-20 days, 20-30 days, 20-25 days, or 25-30 days post administration ofthe one or more compositions of the present disclosure.

Improved Traits

In some aspects, the present disclosure is drawn to administeringmicrobial compositions described herein to poultry to improve one ormore desirable traits such as the modulation of aspects of weight,musculature, meat characteristics, egg quantity, egg weight, egg volume,egg quality, egg shell density, digestive chemistry, efficiency of feedutilization and digestibility, fecal output, methane production, overallbird health, prevention of colonization of pathogenic microbes, andclearance of pathogenic microbes. In some aspects, the improvement oftraits includes an improvement of the innate immune response in poultryor eggs of poultry. In some aspects, the improved innate immune responseis an increase or decrease of lysozyme, steroids, avidin, apoprotein,ovomucoid, ovomucin, ovoflavoprotein, ovoinhibitor, or conalbumin in thepoultry or eggs of poultry. In some aspects, the improvement of traitsinclude an increased success in hatching, an increased incidence ofnormal chick morphology, and increased incidence of embryo survival, anincreased growth rate in chicks and or embryos, an increase in totalbody mass in chicks and poultry, and increase or decrease in egg-whiteproteins.

In some aspects, the improvement of one or more desirable traits is animprovement of 2, 3, 4, 5, 6, 7, 8, 9, or 10 desirable traits in poultryby the administration of microbial compositions described herein.

In some embodiments, the increase in egg quantity is an increase of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 eggs relative to an animal nothaving been administered a composition of the present disclosure. Insome embodiments, the increase in egg quantity is an increase of lessthan 2, 3, 4, 5, 6, 7, 8, 9, or 10 eggs relative to an animal not havingbeen administered a composition of the present disclosure. In someembodiments, the increase in egg quantity is an increase of at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%,140%, 150%, 160%, 170%, 180%, 190%, or 200% relative to an animal nothaving been administered a composition of the present disclosure.

In some embodiments, the increase in egg volume is an increase of atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to an animal not havingbeen administered a composition of the present disclosure. In someembodiments, the increase in egg volume is an increase of less than 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% relative to an animal not having beenadministered a composition of the present disclosure.

In some embodiments, the fecal output is reduced by at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to an animal nothaving been administered a composition of the present disclosure. Insome embodiments, the fecal output is reduced by less than 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to an animal nothaving been administered a composition of the present disclosure.

In some embodiments, the poultry having been administered a compositionof the present disclosure exhibit a weight gain of at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a poultry nothaving been administered a composition of the present disclosure.

In some embodiments, the poultry having been administered a compositionof the present disclosure exhibit a weight gain of at least about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a poultrynot having been administered a composition of the present disclosure.

In some embodiments, the poultry having been administered a compositionof the present disclosure exhibit a feed conversion ratio decrease of atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to apoultry not having been administered a composition of the presentdisclosure.

In some embodiments, the poultry having been administered a compositionof the present disclosure exhibit a feed conversion ratio decrease of atleast about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relativeto a poultry not having been administered a composition of the presentdisclosure.

In some embodiments, the poultry having been administered a compositionof the present disclosure exhibit a decrease in the number of necroticenteritis-causing bacteria in the gastrointestinal tract of at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a poultrynot having been administered a composition of the present disclosure.

In some embodiments, the poultry having been administered a compositionof the present disclosure exhibit a decrease in the number of necroticenteritis-causing bacteria in the gastrointestinal tract of at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to apoultry not having been administered a composition of the presentdisclosure.

In some embodiments, the poultry having been administered a compositionof the present disclosure exhibit a decrease in the number of pathogenicbacteria in the gastrointestinal tract of at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% relative to a poultry not having beenadministered a composition of the present disclosure.

In some embodiments, the poultry having been administered a compositionof the present disclosure exhibit a decrease in the number of humanpathogenic bacteria in the gastrointestinal tract of at least about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a poultrynot having been administered a composition of the present disclosure.

In some embodiments, the poultry having been administered a compositionof the present disclosure exhibit a decrease in the number of poultrypathogenic bacteria in the gastrointestinal tract of at least about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to poultry nothaving been administered a composition of the present disclosure.

In some embodiments, the weight of a flock or flocks of poultry havingbeen administered a composition of the present disclosure exhibit adecrease in the coefficient of variation of at least about 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, %, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a flock of poultry nothaving been administered a composition of the present disclosure.

In some embodiments, the weight of a flock or flocks of poultry havingbeen administered a composition of the present disclosure exhibit anincrease in flock uniformity of at least about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% relative to a flock of poultry not havingbeen administered a composition of the present disclosure.

In some embodiments, the mortality of a flock or flocks of poultryhaving been administered a composition of the present disclosure exhibita decrease in mortality of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% relative to a flock of poultry not having beenadministered a composition of the present disclosure.

In some embodiments, improving the eggs produced by poultry isdesirable, wherein the eggs include triglycerides, triacylglycerides,diacylglycerides, monoacylglycerides, phospholipids, cholesterol,glycolipids, and free fatty acids. In further embodiments, free fattyacids include short chain fatty acids (i.e., C4:0, C6:0, and C8:0),medium chain fatty acids (i.e., C10:0, C10:1, C12:0, C14:0, C14:1, andC15:0), and long chain fatty acids (i.e., C16:0, C16:1, C17:0, C17:1,C18:0, C18:1, C18:2, C18:3, and C20:0).

In some embodiments, improving the quantity or concentration of vitaminsin eggs produced by poultry is desirable. Vitamins found in eggs includeB1, B2, B3, B5, B6, B12, choline, biotin, and folic acid.

In some embodiments, improving the quantity or concentration of mineralsin eggs produced by poultry is desirable. Minerals found in eggs includephosphorous, iodine, selenium, and calcium. Trace amounts of thefollowing may be found in eggs: barium, copper, iron, manganese, nickel,lead, selenium, strontium, vanadium, selenium, rubidium, and zinc.

In some embodiments, increasing or decreasing chicken serum levels ofcalcium, phosphorous, magnesium, triglycerides, cholesterol, andsaccharides is desirable. The modulation of these serum componentsimpact egg traits such as thickness, porosity, density, nutritionalcontent, desirable taste, fat content, cholesterol content, andcoloration.

In some embodiments, improving the efficiency and digestibility ofanimal feed is desirable. In some embodiments, increasing thedegradation of lignocellulosic components from animal feed is desirable.Lignocellulosic components include lignin, cellulose, and hemicellulose.

In some embodiments, increasing the concentration of fatty acids in thegastrointestinal tract is desirable. Fatty acids include acetic acid,propionic acid, and butyric acid. In some embodiments, maintaining thepH balance in the gastrointestinal tract to prevent destruction ofbeneficial microbial bioensembles is desirable. In some embodiments,increasing the concentration of lactic acids in the gastrointestinaltract is desirable. Lactic acid is lowers the pH of the surroundingenvironment, including intracellular pH which can disrupt microbialproton motive force. Lactic acid can also permeabilized the outermembrane of gram-negative bacteria such that they exhibit an increasedsusceptibility to antimicrobials.

In some embodiments, decreasing the amount of methane and manureproduced by poultry is desirable

In some embodiments, a decrease in the amount of total manure producedis desirable. In further embodiments, a decrease in the total amount ofphosphorous and/or nitrogen in the total manure produced is desirable.

In some embodiments, improving the feed intake is desirable. In someembodiments, improving the efficiency of nitrogen utilization of thefeed and/or dry matter ingested by poultry is desirable.

In some embodiments, the improved traits of the present disclosure arethe result of the administration of the presently described microbialcompositions. It is thought that the microbial compositions modulate themicrobiome of poultry such that the biochemistry of one or more elementsof the gastrointestinal tract is changed in such a way that thegastrointestinal liquid and solid substratum are more efficiently andmore completely degraded into subcomponents and metabolites than thegastrointestinal tract of poultry not having been administered microbialcompositions of the present disclosure.

In some embodiments, the increase in efficiency and the increase ofdegradation of the gastrointestinal substratum result in an increase inimproved traits of the present disclosure.

In some embodiments, the improvement of any one or more of the traits ofthe present disclosure is a change of about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%,about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%,about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%,about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%,about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%,about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or about 100% relative to the animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the improvement of any one or more of the traits ofthe present disclosure is a change of at least 0.1%, at least 0.2%, atleast 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%,at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 11%, at least 12%, at least 13%, at least14%, at least 15%, at least 16%, at least 17%, at least 18%, at least19%, at least 20%, at least 21%, at least 22%, at least 23%, at least24%, at least 25%, at least 26%, at least 27%, at least 28%, at least29%, at least 30%, at least 31%, at least 32%, at least 33%, at least34%, at least 35%, at least 36%, at least 37%, at least 38%, at least39%, at least 40%, at least 41%, at least 42%, at least 43%, at least44%, at least 45%, at least 46%, at least 47%, at least 48%, at least49%, at least 50%, at least 51%, at least 52%, at least 53%, at least54%, at least 55%, at least 56%, at least 57%, at least 58%, at least59%, at least 60%, at least 61%, at least 62%, at least 63%, at least64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or at least 100% relative to the animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the increase of any one or more of the traits ofthe present disclosure is an increase of about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%,about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%,about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%,about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%,about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%,about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or about 100% relative to the animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the increase of any one or more of the traits ofthe present disclosure is an increase of at least 0.1%, at least 0.2%,at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, atleast 9%, at least 10%, at least 11%, at least 12%, at least 13%, atleast 14%, at least 15%, at least 16%, at least 17%, at least 18%, atleast 19%, at least 20%, at least 21%, at least 22%, at least 23%, atleast 24%, at least 25%, at least 26%, at least 27%, at least 28%, atleast 29%, at least 30%, at least 31%, at least 32%, at least 33%, atleast 34%, at least 35%, at least 36%, at least 37%, at least 38%, atleast 39%, at least 40%, at least 41%, at least 42%, at least 43%, atleast 44%, at least 45%, at least 46%, at least 47%, at least 48%, atleast 49%, at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 100% relative to the animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the decrease of any one or more of the traits ofthe present disclosure is a decrease of about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%,about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%,about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%,about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%,about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%,about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or about 100% relative to the animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the decrease of any one or more of the traits ofthe present disclosure is a decrease of at least 0.1%, at least 0.2%, atleast 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%,at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 11%, at least 12%, at least 13%, at least14%, at least 15%, at least 16%, at least 17%, at least 18%, at least19%, at least 20%, at least 21%, at least 22%, at least 23%, at least24%, at least 25%, at least 26%, at least 27%, at least 28%, at least29%, at least 30%, at least 31%, at least 32%, at least 33%, at least34%, at least 35%, at least 36%, at least 37%, at least 38%, at least39%, at least 40%, at least 41%, at least 42%, at least 43%, at least44%, at least 45%, at least 46%, at least 47%, at least 48%, at least49%, at least 50%, at least 51%, at least 52%, at least 53%, at least54%, at least 55%, at least 56%, at least 57%, at least 58%, at least59%, at least 60%, at least 61%, at least 62%, at least 63%, at least64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or at least 100% relative to the animal not having beenadministered one or more microbial compositions of the presentdisclosure.

In some embodiments, the villi of the gastrointestinal tract of poultryincrease in diameter and/or length upon administration of one or moremicrobes and/or bioensembles of the present disclosure.

In some embodiments, the villi of the gastrointestinal track of poultryincrease in diameter and/or length upon administration of one or moremicrobes and/or bioensembles of the present disclosure by at least 1 μM,at least 2 μM, at least 3 μM, at least 4 μM, at least 5 μM, at least 6μM, at least 7 μM, at least 8 μM, at least 9 μM, at least 10 μM, atleast 11 μM, at least 12 μM, at least 13 μM, at least 14 μM, at least 15μM, at least 16 μM, at least 17 μM, at least 18 μM, at least 18 μM, atleast 20 μM, at least 21 μM, at least 22 μM, at least 23 μM, at least 24μM, at least 25 μM, at least 30 μM, at least 35 μM, at least 40 μM, atleast 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, at least 65μM, at least 70 μM, at least 75 μM, at least 80 μM, at least 85 μM, atleast 90 μM, at least 95 μM, at least 100 μM, at least 110 μM, at least120 μM, at least 130 μM, at least 140 μM, at least 150 μM, at least 160μM, at least 170 μM, at least 180 μM, at least 190 μM, or at least 200μM.

In some embodiments, the villi of the gastrointestinal track of poultryincrease in diameter and/or length upon administration of one or moremicrobes and/or bioensembles of the present disclosure by less than 1μM, less than 2 μM, less than 3 μM, less than 4 μM, less than 5 μM, lessthan 6 μM, less than 7 μM, less than 8 μM, less than 9 μM, less than 10μM, less than 11 μM, less than 12 μM, less than 13 μM, less than 14 μM,less than 15 μM, less than 16 μM, less than 17 μM, less than 18 μM, lessthan 18 μM, less than 20 μM, less than 21 μM, less than 22 μM, less than23 μM, less than 24 μM, less than 25 μM, less than 30 μM, less than 35μM, less than 40 μM, less than 45 μM, less than 50 μM, less than 55 μM,less than 60 μM, less than 65 μM, less than 70 M, less than 75 μM, lessthan 80 μM, less than 85 μM, less than 90 μM, less than 95 μM, less than100 μM, less than 110 μM, less than 120 μM, less than 130 μM, less than140 μM, less than 150 μM, less than 160 μM, less than 170 μM, less than180 μM, less than 190 μM, or less than 200 μM.

In some embodiments, the ileum or ileal tissue of poultry is thinnerupon administration of one or more microbes and/or bioensembles of thepresent disclosure. The ileal tissue is defined as the tissue of theileam that spans the lumen to the basal membrane.

In some embodiments, the ileum or ileal tissue of poultry is thinnerupon administration of one or more microbes and/or bioensembles of thepresent disclosure by at least 1 μM, at least 2 M, at least 3 μM, atleast 4 μM, at least 5 μM, at least 6 μM, at least 7 μM, at least 8 μM,at least 9 μM, at least 10 μM, at least 11 μM, at least 12 μM, at least13 μM, at least 14 μM, at least 15 μM, at least 16 μM, at least 17 μM,at least 18 μM, at least 18 μM, at least 20 μM, at least 21 μM, at least22 μM, at least 23 μM, at least 24 μM, at least 25 μM, at least 30 μM,at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least55 μM, at least 60 μM, at least 65 μM, at least 70 μM, at least 75 μM,at least 80 μM, at least 85 μM, at least 90 μM, at least 95 μM, at least100 μM, at least 110 μM, at least 120 μM, at least 130 μM, at least 140μM, at least 150 μM, at least 160 μM, at least 170 μM, at least 180 μM,at least 190 μM, or at least 200 μM.

In some embodiments, the ileum or ileal tissue of poultry is thinnerupon administration of one or more microbes and/or bioensembles of thepresent disclosure by less than 1 μM, less than 2 μM, less than 3 μM,less than 4 μM, less than 5 μM, less than 6 μM, less than 7 μM, lessthan 8 μM, less than 9 μM, less than 10 μM, less than 11 μM, less than12 μM, less than 13 μM, less than 14 μM, less than 15 μM, less than 16μM, less than 17 μM, less than 18 μM, less than 18 μM, less than 20 μM,less than 21 μM, less than 22 μM, less than 23 μM, less than 24 μM, lessthan 25 M, less than 30 μM, less than 35 μM, less than 40 μM, less than45 μM, less than 50 μM, less than 55 μM, less than 60 μM, less than 65μM, less than 70 μM, less than 75 μM, less than 80 μM, less than 85 μM,less than 90 μM, less than 95 μM, less than 100 μM, less than 110 μM,less than 120 μM, less than 130 μM, less than 140 μM, less than 150 μM,less than 160 μM, less than 170 μM, less than 180 μM, less than 190 μM,or less than 200 μM.

In some embodiments, the digestibility of fats is increased or improvedin poultry upon the administration of one or more microbes and/orbioensembles of the present disclosure. In some embodiments, ameasurement of crude fat utilized is determined by subtracting the fatin the excreta from the fat in the food eaten.

In some embodiments, the digestibility of fats is increased or improvedin poultry upon the administration of one or more microbes and/orbioensembles of the present disclosure by at least 0.5%, at least 1%, atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95%. In someembodiments, the digestibility of fats is measured by the crude fat inthe excreta subtracted from the crude fat in the food eaten. See Duerret al. (2017. J. Avian Med. Surg. 31(2):132-141.)

In some embodiments, the digestibility of amino acids oroligo-/polypeptides is increased or improved in poultry upon theadministration of one or more microbes and/or bioensembles of thepresent disclosure.

In some embodiments, the digestibility of amino acids oroligo-/polypeptides is increased or improved in poultry upon theadministration of one or more microbes and/or bioensembles of thepresent disclosure by at least 0.5%, at least 1%, at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95%.

In some embodiments, the digestibility of retinol and/or lutein isincreased or improved in poultry upon the administration of one or moremicrobes and/or bioensembles of the present disclosure.

In some embodiments, the digestibility of retinol and/or lutein isincreased or improved in poultry upon the administration of one or moremicrobes and/or bioensembles of the present disclosure by at least 0.5%,at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

In some embodiments, the absorption of calcium, iron, zinc, copper,phosphorous, or ions thereof, is increased or improved in poultry uponthe administration of one or more microbes and/or bioensembes of thepresent disclosure.

In some embodiments, the absorption of calcium, iron, zinc, copper,phosphorous, or ions thereof, is increased or improved in poultry uponthe administration of one or more microbes and/or bioensembes of thepresent disclosure by at least 0.5%, at least 1%, at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95%.

In some embodiments, the occurrence of foamy digesta in thegastrointestinal tract in poultry upon the administration of one or moremicrobes and/or bioensembles of the present disclosure.

In some embodiments, the occurrence of foamy digesta in thegastrointestinal tract in poultry upon the administration of one or moremicrobes and/or bioensembles of the present disclosure by at least 0.5%,at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

In some embodiments, the production of methane in the gastrointestinaltract in poultry is decreased upon the administration of one or moremicrobes and/or bioensembles of the present disclosure.

In some embodiments, the production of methane in the gastrointestinaltract in poultry is decreased upon the administration of one or moremicrobes and/or bioensembles of the present disclosure by at least 0.5%,at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

Mode of Action: Gastrointestinal Health Improvement and CompetitiveExclusion

The influence of the gastrointestinal microbiome on broiler health iswell known (Roberts, 2015; Yeoman, 2012; Lee (presentation); Oakley,2014)—a healthy intestinal system will improve the overall welfare andperformance of birds in a commercial farm setting. Although the exactroles and mechanisms of individual species within this intricate andcomplicated system are still largely unknown, the overall beneficialeffects of microorganisms on the host's health and performance have beenstudied. The current knowledge of metabolism and mechanisms of actionare summarized below. See FIG. 5 . (Pourabedin and Zhao. 2015. FEMSMicrobiol. Lett. 362:fnv122). FIG. 5 depicts a suite of interactionsthat are all modulated by the composition of the gastrointestinal tractwith a well-balanced population of commensal microbes with an adequatesupply of prebiotic compositions. For example, the commensal bacteriaare (1) producing antibacterial compounds to compete with otherorganisms, including pathogens, (2) producing simple fatty acidsinvolved in metabolic regulation and energy use, (3) immunomodulatingthe localized immune responses in conjunction with lymphocytes andantigen presenting cells, etc.

General Nutrition and Gut Health

Increasing the Concentration of Beneficial Molecules, Including ShortChain Fatty Acids and Other Organic Acids, in the Gastrointestinal Tractof the Broiler Improves Bird Performance.

Microbial short chain fatty acid production, in particular, are absorbedand metabolized by the bird and can provide 5% to 15% of the dailyrequirements for bird maintenance energy (Chichlowski, 2007; Annison,1968; Gasaway, 1976ab). Previous studies have shown that supplementationof butyrate can improve both overall weight gain and feed-conversionwhen administered daily to the bird, and that supplementation of anyorganic acid (including fumaric and lactic) can improve bird weight gain(Levy, 2015; Gilliland, 1977; Afil, 2010). Levy, et al. (2015) showedthat improvements in body weight gain and feed conversion increasedlinearly with increasing concentrations of encapsulated butyric acidlevels. Butyrate also enhances vili development (Chamba, 2014) activatesthe immune response, and can also have a direct bactericidal effect(Gantois, 2006).

Improving Development of the Gastrointestinal Tract, Enhancing VilliGrowth, and Stimulating the Immune System.

Supplementation of butyrate and other organic acids to the diets ofbirds have been shown to enhance vili development and stimulate theimmune system (Chamba, 2014; Adil 2010; Adil 2011).

Improving Apparent Metabolizable Energy of the Diet

Fermentation of various microbes can convert carbohydrates to variousend products. Most short chain fatty acids produced by thesemicroorganisms are absorbed and utilized by the bird (Rinttila, 2013;Annison, 1968; Gasaway, 1976ab). The synthesis of vitamins, includingvitamins B and K, are also carried out by microorganisms (Cummings,1997).

Competitive Exclusion

Bacteriocin Production

Microorganisms within the gastrointestinal tract self-regulate throughthe production of various antimicrobial chemicals. Bacteriocins, forexample, are commonly produced by lactic acid microorganisms and canprevent the colonization of pathogens (Chen, 2007; Juven 1990).Short-chain fatty acids been shown to impact and inhibit entericbacteria including Salmonella typhimurium, but do not inhibitbeneficial, native microorganisms (Van der Wielen et al., 2000). Bothpropionic acid, butyric acid, acetate has also been shown to inhibitpathogenic bacteria (Marounek, 1999; Van der Wielen, 2000; Immerseei,2003).

Competitive Use of Nutrients/Binding Sites

Birds are first inoculated with microorganisms shortly after birth. Asthe bird continues to develop, the microbiome colonizes and establishesitself, ultimately creating a stable ecosystems that houses organismsthat occupy all niches and utilizes all available nutrients (Callaway,2008). This expansive, stable community can prevent pathogens fromcolonizing.

Creating Environments that are not Conducive to Pathogen Growth

Microorganisms residing within the gut reduce the redox potential withinthe gut, creating an environment suitable for obligate anaerobes toflourish (Cummings, 1997; Chicklowki, 20017; Juven 1990). Lactate andother short chain fatty acid production lowers the pH of thegastrointestinal environment, making it more difficult for pathogens tocolonize and grow (Pourabedin, 2015). Native microorganisms have alsobeen shown to neutralize enterotoxins (M'Sadeq, 2015).

EXAMPLES Example I. Administration and Colonization of Native BacteriaFed to Young Broiler Chicks

Purpose: Determine the colonization efficacy of native microorganismsfed to broiler chicks at varying times during development.

Procedure: Ascusbbr_105932, Ascusbbr_10593, and Ascusbbr_2676 wereadministered to chicks. A total of 60 chicks were evenly divided into 3groups of 20 birds. Each group of 20 birds were housed in an independentbattery cage with at least one empty cage in between the groups to avoidcross contamination. The three groups were: experimental group 1,experimental group 2, and the control group.

Experimental group 1 received 0.5 mL of a microbe composition containingAscusbbr_10593, Asucsbbr_2676, and Ascusbbr_105932 suspend in 1×RAMMsaline solution via gavage on day 0 of age. Experimental group 2received 0.5 mL of a microbe composition containing Ascusbbr_10593,Asucsbbr_2676, and Ascusbbr_105932 suspended in 1×RAMM saline solutionon day 5 of age via gavage. The control group received 0.5 mL of sterile1×RAMM saline solution via gavage. Experimental group 1 and experimentalgroup 2 received approximately 1×10⁶ cells of each strain. On day 7, day11, day 14, and day 18 of age, 4 birds from each group were randomlyselected and removed from the cages to be sampled. Each bird had thefollowing organs sampled: cecum, small intestine content, and smallintestine scraping via swab. For cecum and small intestine content thesamples were collected in a 2 mL tube containing 200 μL of stop solution(95% ethanol and 5% phenol). For the swabs, the 2 mL collection tubeswere prefilled with 600 μL of a stop solution-PBS mixture (25% of stopsolution (95% ethanol and 5% phenol) and 75% 1× sterile PBS). Swabs weredipped into the stop solution-PBS mixture and then placed into a new,empty 2 mL collection tube for storage. The samples were then stored at−20° C. until shipment and processing.

Nucleic acids were extracted from each sample. Both DNA and RNA wereamplified using PCR, and the libraries were prepped for Illumina MiSeqsequencing. After sequencing, the 16S data was analyzed using USEARCHsoftware, and the administered strains were identified in both theexperimental groups and the control group. Table 12, Table 13, and Table14 show the increases and decreases in the abundance of each straincompared to the control group.

Results:

TABLE 12 Changes in abundance of the administered strains in the cecumcontent of the broilers Day 11 Day 14 Group Ascusbbr_105932Ascusbbr_10593 Ascusbbr_2676 Ascusbbr_105932 Ascusbbr_10593 ExperimentalGroup 1: − − − − + Day 0 admin Experimental Group 2: − + − − + Day 5admin Day 14 Day 18 Group Ascusbbr_2676 Ascusbbr_105932 Ascusbbr_10593Ascusbbr_2676 Experimental Group 1: − − − − Day 0 admin ExperimentalGroup 2: − − − − Day 5 admin

Day 7 data was omitted because samples had <100 total reads. ‘−’represents the relative abundance for that sample being lower than orequal to the relative abundance of the control. ‘+’ represents therelative abundance being greater than the relative abundance of thecontrol sample.

TABLE 13 Changes in abundance of the administered strains in the smallintestinal content of the broilers Day 7 Day 11 Group Ascusbbr_105932Ascusbbr_10593 Ascusbbr_2676 Ascusbbr_105932 Ascusbbr_10593Ascusbbr_2676 Experimental Group 1: − − − − + − Day 0 admin ExperimentalGroup 2: − + − − − − Day 5 admin Day 14 Day 18 Group Ascusbbr_105932Ascusbbr_10593 Ascusbbr_2676 Ascusbbr_105932 Ascusbbr_10593Ascusbbr_2676 Experimental Group 1: − − − − − + Day 0 admin ExperimentalGroup 2: − − − − − + Day 5 admin

‘−’ represents the relative abundance for that sample being lower thanor equal to the relative abundance of the control. ‘+’ represents therelative abundance being greater than the relative abundance of thecontrol sample.

TABLE 14 Changes in abundance of the administered strains in the smallintestinal wall scraping of the broilers Day 7 Day 11 GroupAscusbbr_105932 Ascusbbr_10593 Ascusbbr_2676 Ascusbbr_105932Ascusbbr_10593 Ascusbbr_2676 Experimental Group 1: − − − − − − Day 0admin Experimental Group 2: − + + − − + Day 5 admin Day 14 Day 18 GroupAscusbbr_105932 Ascusbbr_10593 Ascusbbr_2676 Ascusbbr_105932Ascusbbr_10593 Ascusbbr_2676 Experimental Group 1: − − − − − − Day 0admin Experimental Group 2: − − + − − − Day 5 admin

‘−’ represents the relative abundance for that sample being lower thanor equal to the relative abundance of the control. ‘+’ represents therelative abundance being greater the relative abundance of the controlsample.

Discussion:

Ascusbbr_105932 failed to colonize in any of the organs that were testedacross all time points. Ascusbbr_10593 seems to establish at a higherabundance than the control in the cecum of experimental group 2. In thececum of group 2, there was establishment on day 11 and day 14 of age,while on day 18 of age the relative abundance was equal to or less thanthe relative abundance of the control group. The increase in abundanceof Ascusbbr_10593 showed that there was increased colonization ofAscusbbr_10593 as compared to the control. Without continuousadministration of Ascusbbr_10593, the microbiota of the cecum returnedto a more natural state by day 18 of age. This trend has been shown inmultiple animals and studies where there was an attempt to change thenatural microbiota. See Preidis et al. (2012. FASEB J. 26:1960-1969),Weimer et al. (2010. J. Dairy Sci. 93:5902-5912), Weimer (2015. Front.Microbiol. 6:1-16), and Weimer et al. (2017. J. Dairy Sci.100:7165-7182). Ascusbbr_10593 also showed colonization on day 7 of ageof the small intestine scrapings in group 2 before the microbiota returnto its natural state on day 11 of age. The results of the smallintestine scrapings are also mirrored in the small intestine content,showing that there is greater colonization of Ascusbbr_10593 when thechicks were gavaged with at day 5 of age.

Ascusbbr_2676 in experimental group 2 colonized in the small intestinescrapings on day 7 and day 11 of age before it returned to a similarmicrobiota of the control on day 18 of age. The lack of Ascusbbr_2676colonization in the small intestine content suggests that Ascusbbr_2676binding to the mucosal layer through collagen (types-III, IV, and V),gelatin, fibrinogen, lectin, laminin, or vitronectin.

For experimental group 1, where microbes were administered on day 0 ofage, there seems to be inconsistent results and lack of evidence ofAscusbbr_10593, Ascusbbr_2676, and Ascusbbr_105932 colonization. Thiscould be due to the anti-microbial properties that a young chickpossesses early in its life span within the GI tract. Administration ofmicrobes on day 5 of age compared to day 0 of age seems to benefit theestablishment and colonization of Ascus microbes.

REFERENCES

-   Preidis, G. A. et al. Probiotics stimulate enterocyte migration and    microbial diversity in the neonatal mouse intestine. FASEB J. 26,    1960-1969 (2012).-   Weimer, P. J., Stevenson, D. M., Mantovani, H. C. & Man, S. L. C.    Host specificity of the ruminal bacterial community in the dairy cow    following near-total exchange of ruminal contents. J. Dairy Sci. 93,    5902-5912 (2010).-   Weimer, P. J. Redundancy, resilience, and host specificity of the    ruminal microbiota: Implications for engineering improved ruminal    fermentations. Front. Microbiol. 6, 1-16 (2015).-   Weimer, P. J. et al. Transient changes in milk production efficiency    and bacterial community composition resulting from near-total    exchange of ruminal contents between high- and low-efficiency    Holstein cows. J. Dairy Sci. 100, 7165-7182 (2017).

Example II. Lectin-Binding Testing of Broiler Isolates

Immediately after hatching, broiler chicks are protected from pathogensby the antimicrobial properties of egg whites. See Hager et al. (1983.Poultry Science. 62:247-254). After this protection disappears, chicksare constantly challenged with pathogens and never converge on a coremicrobiome. This leads to high death rates due to necrotic enteritis andchronically inflamed intestinal epithelial. Through competitiveexclusion via the founder effect, broiler strains that display collagenand mucin binding have the ability to exclude pathogens. This has beenshown in many cases to increase overall immune health and decrease thechronically inflamed intestinal epithelial. See Yadav et al. (2013.Microbiol. Res. 168:639-645). Here, we test 4 strains (strain 1, strain2, positive control, and negative control) for the ability to bindparticular lectins using ELISAs.

The lectin binding assays are designed to identify phenotypic mannose,fucose and N-acetylglucosamine lectin binding via a Sandwich ELISA. Eachstrain is tested in triplicate and compared to positive and negativecontrols. The binding is a calcium-dependent lectin binding proteininvolved in immune defense. The ELISA sandwich assay utilizes theantibody specific for chicken mannose-binding lectin. There are manysynonyms to this type of binding including but not limited toCollectin-1, Mannan-binding protein, Mannose-binding lectin,Mannose-binding protein C, MBL2, MBL2_CHICKEN, MBP-C, MBP1. Furthermore,this assay tests for both bacteria that contain both 1 C-type lectindomain and 1 collagen-like domain.

Standard Preparation

Serially diluted standards are prepared fresh prior to use. Broiler MBLstandard is used as a positive control in combination with broilerchicken serum/plasma samples. Tubes are mixed gently. 25 ng/mL standardsare serially diluted at 200 ul mixing gently at each dilution. Blankcontrols contain no protein.

Sample and Plate Preparation

All isolated are grown to sufficient turbidity and pelleted throughspinning. All samples are split to create technical triplicates fortesting, with the suggested dilution for normal serum/plasma: 800 fold.

Assay Protocol

All materials are equilibrated to room temperature all samples andstandards are performed in triplicates. 100 uL of each sample andstandard are pipetted into the appropriate wells. The plate is coveredand incubated at room temperature overnight at 4 degrees C. Afterincubation samples are washed 4 times with PBS wash buffer and removedthrough aspiration. 100 ul of prepared biotinylated Human MBL DetectionAntibody and then incubated for one hour at room temperature with gentleshaking. 100 ul of 1×HRP-Streptavidin solution is added to each well andfor 45 minuets at room temperature with gentle shaking. 100 ul of TMB isadded to each well and for 30 minuets at room temperature with gentleshaking. Finally, 50 μL of Stop Solution is added to each well. Opticaldensity at 450 nm is read immediately following.

Anti-Inflammatory to In Vivo Test

Organisms that test positive for binding domains are grown and tested invivo in broiler chickens through an oral gavage. Broilers are tested forSerum α-1-AGP, CD4+ lymphocytes, CD8+ lymphocytes, cytokines IL8 andTNFSF15, IL17F, iNOS expression, and Gene Expression of tight junctionproteins JAM2, occludin, ZO1, and MUC2. See Gadde et al. (2017. Res. VetSci. 114:236-243).

Results

Testing of binding assay on strain isolates one and two will likelyreveal clear binding in only isolate two (FIG. 6 ). Isolate two is thenused to orally gavage broiler chickens. Serum from gavaged chickens areconfirmed to be immunologically improved through Serum α-1-AGP, CD4+lymphocytes, CD8+ lymphocytes, cytokines IL8 and TNFSF15, IL17F, iNOSexpression, and Gene Expression of tight junction proteins JAM2,occludin, ZO1, and MUC2 will be compared to control serum with a p-valueof 1e-16.

REFERENCES

-   Hager J E, Beane W L. 1983. Posthatch incubation time and early    growth of broiler chickens. Poult Sci 62:247-254.-   Yadav A K, Tyagi A, Kaushik J K, Saklani A C, Grover S, Batish    V K. 2013. Role of surface layer collagen binding protein from    indigenous Lactobacillus plantarum 91 in adhesion and its    anti-adhesion potential against gut pathogen. Microbiol Res    168:639-645.-   Gadde U D, Oh S, Lee Y, Davis E, Zimmerman N, Rehberger T, Lillehoj    H S. 2017. Dietary Bacillus subtilis-based direct-fed microbials    alleviate LPS-induced intestinal immunological stress and improve    intestinal barrier gene expression in commercial broiler chickens.    Res Vet Sci 114:236-243.

Example III. Collagen-Binding Testing of Broiler Isolates

It has long been established that Clostridium perfringens is a majorcausative agent of necrotic enteritis, a common poultry diseaseestimated to cost the world $6 billion a year. See Immerseel et al.(2009. Trends Microbiol. 17:32-36) and Wade and Keyburn (2015. WorldPoultry. 31(7):16-17). “The ability to adhere to the host's intestinalepithelium and to extracellular matrix molecules in the gut are wellknown strategies used by bacterial enteropathogens,” and it ispostulated that C. perfringens uses a similar strategy to colonizepoultry intestinal cells through the binding of extracellular matrixmolecules including collagen. See Martin and Smyth (2010. Anaerobe.16(5):533-539). Various studies have illustrated a strong correlationbetween C. perfringens' ability to adhere to various collagen types andthe strain's virulence. See Wade et al. (2016. Vet. Microbiol.197:53-61). Therefore, a collagen binding assay is designed with thepurpose of discovering microorganisms that could competitively inhibitC. perfringens colonization by preferentially binding select collagenmotifs that are also adhered to by C. perfringens. Our results will showthat our avirulent strain is able to bind collagen types III through Vand gelatin with similar or greater affinity than virulent C.perfringens.

This assay based upon a protocol designed by Wade et al., investigatingthe binding of various bacteria to gelatin and collagen types II, III,IV, V. See Wade et al. (2015. Vet. Microbiol. 180(3-4):299-303).

Solubilizing Collagen and Gelatin

The following stocks are created: 1 mg of collagen type II and IV isadded to 50 ml PBS and 0.13 ml acetic acid and shaken overnight at 4° C.1 mg of collagen type III is added to 48.5 ml PBS and 1.5 ml acetic acidand shaken overnight at 4° C. 1 mg of collagen type V is added to 48.8ml PBS and 1.2 ml of acetic acid and shaken at room temp for 3 hours.Gelatin is added to 49.7 ml PBS and 0.3 ml acetic acid and shaken atroom temp for 3 hours.

Collagen and Gelatin Adherence

50 ul of the 1 mg/50 ml solutions of either: collagen type II, III, IV,V, gelatin, or PBS is added to each well of a 96 well NUNCLON Deltasurface treated plate. The plates are then incubated overnight in thedark at 4° C.

Blocking the Wells

A blocking solution is made of Ig of BSA and 20 ul of Tween 20 in 40 mlof PBS. 200 ul of this blocking solution is added to each well of theplate. The plate is incubated for 2 hours in the dark at 4° C. Afterthis each well is rinsed three times with 200 ul of PBS.

Cellular Adhesion

A virulent C. perfringens positive for the cnaA gene, an avirulentgram-positive strain positive for the cnaA gene, and a gram-positivestrain negative for the cnaA gene is grown up to late exponential phasein anaerobic TSB+5% defibrinated sheep's blood. 40 ml from thesecultures are then centrifuged at 4,300 RCF for 30 minutes at 4° C.Pellets are then washed three times with fresh PBS and cell suspensionsare adjusted to an optical density of 0.8 at 600 nm. 50 ul of cellsuspension is added to each well and the plate is incubated at roomtemperature for 2 hours in the dark with agitation. After this, eachwell is washed three times with 100 ul of fresh PBS.

Crystal Violet Stain

A solution of 0.5% (w/v) of crystal violet in PBS is made. 100 ul ofthis is added to each well and incubated at room temp in the dark for 5minutes. Then each well is washed three times with 100 ul of fresh PBS.Cells are then destained with 50 ul of 1:1 ethanol:acetone (v/v) andabsorbance of each well is read at 562 nm.

Results

All reported results are corrected by their respective controls. Fromour results, we expect to see that both cnaA positive strains C.perfringens and Strain 1 have a higher binding affinity for all of thetested proteins than the cnaA negative Strain 2. (FIG. 7 ) This is inagreement with previous data 3-5 which shows a correlation between thepresence of the cnaA gene and a strain's ability to bind collagen.Furthermore, these results will indicate that Strain 1 has a greateradherence to gelatin and collagen III, IV and V than C. perfringens.Because of this it is possible that Strain 1 could be used as acompetitive inhibitor to C. perfringens binding of extracellular matrixmolecules in broiler. By inhibiting the colonization of C. perfringens,Strain 1 could reduce the incidence of necrotic enteritis and improvethe health of broiler chickens.

REFERENCES

-   Van Immerseel, F., Rood, J. I., Moore, R. J., Titball, R. W., 2009.    Rethinking our understanding of the pathogenesis of necrotic    enteritis in chickens. Trends Microbiol. 17, 32-36.-   Wade, B., Keyburn, A. L., 2015. The true cost of necrotic enteritis.    World Poultry 31 (7), 16-17.-   Thomas G. Martin, Joan A. Smyth, The ability of disease and    non-disease producing strains of Clostridium perfringens from    chickens to adhere to extracellular matrix molecules and Caco-2    cells, In Anaerobe, Volume 16, Issue 5, 2010, Pages 533-539, ISSN    1075-9964, https://doi.org/10.1016/j.anaerobe.2010.07.003.-   Ben Wade, Anthony L. Keyburn, Volker Haring, Mark Ford, Julian I.    Rood, Robert J. Moore, The adherent abilities of Clostridium    perfringens strains are critical for the pathogenesis of avian    necrotic enteritis, In Veterinary Microbiology, Volume 197, 2016,    Pages 53-61-   B. Wade, A. L. Keyburn, T. Seemann, J. I. Rood, R. J. Moore, Binding    of Clostridium perfringens to collagen correlates with the ability    to cause necrotic enteritis in chickens, In Veterinary Microbiology,    Volume 180, Issues 3-4, 2015, Pages 299-303.

Example III. Collagen-Binding Testing of Broiler Isolates

Purpose: The objective of this study is to test the ability offormulated microbes to colonize young chicks when mixed into mash feedfor 35 days.

Procedure: Ascusbbr_105932, Ascusbbr_5796, and Ascusbbr_2676 wereadministered to chicks. A total of 192 chicks were evenly divided into 4treatment groups. Each treatment group consisted of 3 pens, with eachpen containing 16 birds The four groups were: unchallenged control,positive control (all 3 microbes administered via gavage), experimentalgroup 1, and experimental group 2.

Experimental group 1 received a microbial composition containingAscusbbr_105932, Ascusbbr_2676, and a carrier mixed into feed daily.Experimental group 2 received a microbial composition containingAscusbbr_105932, Ascusbbr_5796, Ascusbbr_2676, and a carrier mixed intofeed daily. Experimental group 1 and experimental group 2 receivedapproximately 1×106 cells of each strain. The positive control receivedAscusbbr_105932, Ascusbbr_5796, and Ascusbbr_2676 suspended in 1×RAMMsaline solution once on day 5 of age via gavage. The control groupreceived the carrier daily via feed. On day 2, day 7, day 14, day 21,day 28, and day 35 of age, 2 birds from each group were randomlyselected and removed from the cages to be sampled. Each bird had thefollowing organs sampled: cecum, small intestine content, and smallintestine scraping via swab. For cecum and small intestine content thesamples were collected in a 2 mL tube containing 200 μL of stop solution(95% ethanol and 5% phenol). For the swabs, the 2 mL collection tubeswere prefilled with 600 μL of a stop solution-PBS mixture (25% of stopsolution (95% ethanol and 5% phenol) and 75% 1× sterile PBS). Swabs weredipped into the stop solution-PBS mixture and then placed into a new,empty 2 mL collection tube for storage. The samples were then stored at−20° C. until shipment and processing.

Nucleic acids were extracted from each sample. Both DNA and RNA wereamplified using PCR, and the libraries were prepped for Illumina MiSeqsequencing. After sequencing, the 16S data was analyzed using USEARCHsoftware, and the administered strains were identified in both theexperimental groups and the control group. Table 12, Table 13, and Table14 show the increases and decreases in the abundance of each straincompared to the control group.

Results: Ascusbbr_105932 showed colonization on the small intestinelining by day 7 in experimental group 1, experimental group 2, and thepositive control. It was not detected in the unchallenged control.

Ascusbbr_2676 showed colonization in experimental group 1, experimentalgroup 2, and the positive control in the small intestine content and onthe small intestine lining. It was not detected in the unchallengedcontrol.

Ascusbbr_5796 showed colonization in experimental group 1, experimentalgroup 2, and the positive control by day 7 in the small intestinecontent and lining. Its abundance was only detected in the unchallengedcontrol group in the small intestine lining samples.

See FIG. 8 for the depiction of the rate of microbial convergence acrossthe experimental groups. Here, day 35 is assumed to represent the finalmicrobiome of the flock. The distance between the microbiome throughoutthe experiment was compared to the average, final, day 35 microbiome ofthe birds. Birds receiving microbes, notably experimental group 2 (3microbes in feed) and the positive control (oral gavage, 3 microbes)displayed a faster convergence to the final microbiome than experimentalgroup 1 and the unchallenged control.

See FIG. 9 for the final percentage mortality of the experiment. Allgroups that received microorganisms (positive control, experimentalgroup 1, and experimental group 2) exhibited lower percentage mortalitythan the control.

Discussion: Here, native microbes were successfully delivered to thegastrointestinal tract of broiler chickens in a stabilized form viafeed. Colonization patterns of the primary target microorganisms weresimilar to the unstabilized microorganisms utilized in the positivecontrol. Ascusbbr_105932 and Ascusbbr_2676 were only detected in thethree groups that received microbes. They were not detected in theunchallenged control. Ascusbbr_5796, however, did appear in all 4experimental groups despite only being administered to experimentalgroup 2 and the positive control. In the 3 groups that receivedmicrobes, Ascusbbr_5796 was detected in the small intestine content andlining. In the unchallenged control, it was only detected in the smallintestine lining. The appearance of Ascusbbr_5796 in all 4 treatmentgroups is unsurprising, as it is a common member of the native broilerchicken microbiome.

Administration of microorganisms was also found to improve theperformance of the birds. All three groups that received microorganismsexhibited lower percent mortality than the unchallenged control.Similarly, the two groups that received three microorganisms(experimental group 2 and the positive control), exhibited a faster rateof microbiome maturity than experimental group 1 and the unchallengedcontrol. A more mature microbiome is more resilient to pathogens andinfection, so these birds would be more likely to survive an outbreak ofa bacterial infection.

TABLE 15 Budapest Treaty Deposits of the Disclosure Depository AccessionNumber Date of Deposit ATCC PTA-124016 Mar. 2, 2017 ATCC PTA-124039 Mar.10, 2017 Bigelow PATENT201703001 Mar. 17. 2017 Bigelow PATENT201703002Mar. 24, 2017 Bigelow PATENT201703003 Mar. 24, 2017 BigelowPATENT201703004 Mar. 24, 2017 NRRL B-67264 May 16, 2016 NRRL B-67265 May16, 2016 NRRL B-67266 May 16, 2016 NRRL B-67267 May 16, 2016 NRRLB-67268 May 16, 2016 NRRL B-67269 May 16, 2016 NRRL B-67270 May 16, 2016NRRL B-67289 Sep. 26, 2018 NRRL B-67290 Sep. 26, 2018 NRRL B-67291 Sep.26, 2018 NRRL B-67292 Sep. 26, 2018

Numbered Embodiments of the Disclosure

Notwithstanding the appended claims, the disclosure sets forth thefollowing numbered embodiments:

1. A method for stimulating the production of B cells, stimulating theproduction of T cells, increasing the length of villi, reducing theexpression of inflammatory cytokines of poultry, the method comprising:

-   -   a) administering to a poultry an effective amount of a poultry        supplement comprising:    -   i) a purified microbial population that comprises a bacterium        with a 16S nucleic acid sequence, and/or a fungus with an ITS        nucleic acid sequence, which is at least about 97% identical to        a nucleic acid sequence selected from the group consisting of        SEQ ID NOs:1-387, and said bacterium and/or fungus have a MIC        score of at least about 0.2; and    -   ii) a carrier suitable for poultry administration,    -   wherein the poultry administered the effective amount of the        poultry supplement exhibits an increase in production of B        cells, an increase in production of T cells, an increase in        length of villi, or a decrease in the expression of inflammatory        cytokines, as compared to poultry not having been administered        the supplement.        2. The method of embodiment 1, wherein the poultry is a broiler.        3. The method according to embodiment 1, wherein the poultry        supplement is stable under ambient conditions for at least one        week.        4 The method according to embodiment 1, wherein the poultry        supplement is formulated as an: encapsulation, tablet, capsule,        pill, feed additive, food ingredient, food additive, food        preparation, food supplement, water additive, water-mixed        additive, heat-stabilized additive, moisture-stabilized        additive, consumable solution, consumable spray additive,        consumable solid, consumable gel, injection, suppository,        drench, or combinations thereof.        5. The method according to embodiment 1, wherein the poultry        supplement is encapsulated in a polymer or carbohydrate.        6, The method according to embodiment 1, wherein the poultry        supplement comprises a glassy matrix comprising the bacterium        and/or the fungus.        7. The method according to embodiment 1, wherein the poultry        supplement exhibits a water activity of less than 0.25.        8. The method according to embodiment 1, wherein the poultry        supplement is administered one or more times at least one day        post-hatching.        9. The method according to embodiment 1, wherein the poultry        supplement is administered one or more times between the day of        hatching and 14 days post-hatching.        10. The method according to embodiment 1, wherein the poultry        supplement comprises a first microbial composition and a second        microbial composition.        11. The method according to embodiment 10, wherein the first        microbial composition is administered daily beginning at day 4        or 5 post-hatching, and the second microbial composition is        administered daily beginning with the first feed change.        12. The method of embodiment 11, wherein the first microbial        composition is no longer administered once the administration of        the second microbial composition begins.        13. The method according to embodiment 1, wherein the poultry        supplement comprises:    -   a) a first microbial composition administered one or more times        between the day of hatching, and    -   b) a second microbial composition administered one or more times        between 15 days post-hatching and harvest of the poultry.        14. The method according to embodiment 1, wherein administering        comprises: feeding the poultry supplement to the poultry.        15. The method according to embodiment 1, wherein administering        comprises: spraying the poultry supplement onto a poultry.        16. The method according to embodiment 1, wherein the purified        microbial population is present in the poultry supplement at a        concentration of at least 102 cells.        17. The method of embodiment 1, wherein the poultry exhibit an        increase in production of B cells.        18. The method of embodiment 17, wherein the B cells are        selected from the group consisting of B-1 cells, B-2 cells,        marginal zone B cells, follicular B cells, memory B cells,        plasma cells, and plasmablasts; or combinations thereof.        19. The method of embodiment 1, wherein the poultry exhibit an        increase in production of T cells.        20. The method of embodiment 19, wherein the T cells are        selected from the group consisting of gamma delta T cells, alpha        beta T cells, natural killer T cells, regulatory T cells, memory        T cells, cytotoxic T cells helper T cells, and effector T cells;        or combinations thereof.        21. The method of embodiment 1, wherein the poultry exhibit an        increase in length of villi.        22. The method of embodiment 21, wherein the villi length        increase by at least 1 μM.        23. The method of embodiment 22, wherein the villi length        increase by less than 10 μM.        24. The method of embodiment 13, wherein the second microbial        composition elicits a thinning of the ileal tissue, a reduction        in pH in the ileum, and/or a reduction in gas production in the        gastrointestinal tract of the poultry.        25. The method of embodiment 11, wherein the second microbial        composition elicits a decrease in the variability of the number        of unique species in the gastrointestinal tract of the poultry.        26. The method of embodiment 24, wherein the poultry exhibit a        thinning of the ileal tissue.        27. The method of embodiment 26, wherein the ileal tissue is        thinned by at least 1 μM.        28. The method of embodiment 26, wherein the ileal tissue is        thinned by less than 200 μM.        29. The method of embodiment 24, wherein the poultry exhibit a        reduction in pH in the ileum by at least 0.6 pH.        30. The method of embodiment 24, wherein the production of gas        in the gastrointestinal tract of the poultry is a reduction of        at least 5%.        31. The method of embodiment 25, wherein the decrease in the        variability of the number of unique species is a reduction of        the total number of unique species of microbes in the small        intestine to between about 100 and 400 species.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following Claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as, an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

1. A method for improving one or more desirable traits in fowl, themethod comprising: (a) administering to a fowl an effective amount of amicrobial composition comprising: (i) a purified microbial populationthat comprises one or more bacteria with a 16S nucleic acid sequencethat shares at least 97% sequence identity with a nucleic acid sequenceselected from SEQ ID NOs: 1-50 and 59-387 and/or one or more fungi withan ITS nucleic acid sequence that shares at least 97% sequence identitywith a nucleic acid sequence selected from SEQ ID NOs: 51-58, and (ii) acarrier suitable for fowl administration; wherein the purified microbialpopulation is present in the microbial composition in an amounteffective to improve the one or more desirable traits as compared to afowl not having been administered the microbial composition; and whereinthe improvement of the one or more desirable traits is an improvement inthe innate immune response, an improvement in incidence of normalgastrointestinal morphology, an improvement in growth rate, animprovement in total body mass, an improvement in feed conversion ratio,an improvement in pathogen exclusion, an improvement in competitiveexclusion against pathogens, a reduction in mortality, a reduction inflock variability, an improvement in antimicrobial production, animprovement in stimulating the production of B cells, an improvement instimulating the production of lymphocytes, an improvement in length ofvilli, and/or an improvement in expression of inflammatory cytokines. 2.The method of claim 1, wherein the fowl is a broiler.
 3. The method ofclaim 1, wherein the one or more bacteria and/or the one or more fungihave a MIC score of at least about 0.1.
 4. The method of claim 1,wherein the purified microbial population comprises a bacterium with a16S nucleic acid sequence sharing at least 97% identity with SEQ IDNO:386.
 5. The method of claim 1, wherein the purified microbialpopulation comprises a bacterium with a 16S nucleic acid sequencesharing at least 97% identity with SEQ ID NO:387.
 6. The method of claim4, wherein the purified microbial population further comprises one ormore bacteria with a 16S nucleic acid sequence sharing at least 97%sequence identity with nucleic acid sequences selected from the groupconsisting of SEQ ID NO:13, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:31, SEQID NO:369, SEQ ID NO:374, and SEQ ID NO:348.
 7. The method of claim 6,wherein the purified microbial population comprises one or more bacteriawith a 16S nucleic acid sequence sharing at least 97% sequence identitywith SEQ ID NO:386 and SEQ ID NO:387.
 8. The method of claim 7, whereinthe purified microbial population comprises one or more bacteria with a16S nucleic acid sequence comprising SEQ ID NO:386 and SEQ ID NO:387. 9.The method of claim 6, wherein the purified microbial populationcomprises one or more bacteria with a 16S nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:13, SEQ ID NO:1, SEQ ID NO:3, SEQID NO:31, SEQ ID NO:369, SEQ ID NO:374, and SEQ ID NO:348.
 10. Themethod of claim 4, wherein the purified microbial population furthercomprises one or more bacteria with a 16S nucleic acid sequence sharingat least 97% sequence identity with nucleic acid sequences selected fromthe group consisting of SEQ ID NO:1 and SEQ ID NO:3.
 11. The method ofclaim 10, wherein the purified microbial population comprises one ormore bacteria with a 16S nucleic acid sequence sharing at least 97%sequence identity with SEQ ID NO:386 and SEQ ID NO:387.
 12. The methodof claim 11, wherein the purified microbial population comprises one ormore bacteria with a 16S nucleic acid sequence comprising SEQ ID NO:386and SEQ ID NO:387.
 13. The method of claim 10, wherein the purifiedmicrobial population comprises one or more bacteria with a 16S nucleicacid sequence selected from the group consisting of SEQ ID NO:1 and SEQID NO:3.
 14. The method of claim 1, wherein the microbial composition isformulated as an encapsulation, tablet, capsule, pill, feed additive,food ingredient, food preparation, food supplement, water additive,water-mixed additive, heat-stabilized additive, moisture-stabilizedadditive, pre-pelleted feed additive, pelleted feed additive,post-pelleting-applied feed additive, consumable solution, consumablespray additive, consumable solid, consumable gel, injection,suppository, drench, bolus, or combination thereof.
 15. The method ofclaim 1, wherein the improvement of the one or more desirable traits isan improvement in antimicrobial production.
 16. The method of claim 15,wherein the improvement of antimicrobial production is an improvement ofantimicrobial production in the gastrointestinal tract.
 17. The methodof claim 1, wherein the administering a purified microbial populationcomprises administering a first microbial composition and a secondmicrobial composition.
 18. The method of claim 1, wherein the microbialcomposition elicits a decrease in the variability of the number ofunique species in the gastrointestinal tract.
 19. The method of claim18, wherein the decrease in the variability of the number of uniquespecies is a reduction of the total number of unique species of microbesto between about 50 and 400 species.
 20. A microbial compositioncomprising: (i) a purified microbial population that comprises one ormore bacteria with a 16S nucleic acid sequence that shares at least 97%sequence identity with a nucleic acid sequence selected from SEQ ID NOs:1-50 and 59-387 and/or one or more fungi with an ITS nucleic acidsequence that shares at least 97% sequence identity with a nucleic acidsequence selected from SEQ ID NOs: 51-58, and (ii) a carrier suitablefor fowl administration; wherein the purified microbial population ispresent in the microbial composition in an amount effective to improvethe one or more desirable traits as compared to a fowl not having beenadministered the microbial composition; and wherein the improvement ofthe one or more desirable traits is an improvement in the innate immuneresponse, an improvement in incidence of normal gastrointestinalmorphology, an improvement in growth rate, an improvement in total bodymass, an improvement in feed conversion ratio, an improvement inpathogen exclusion, an improvement in competitive exclusion againstpathogens, a reduction in mortality, a reduction in flock variability,an improvement in antimicrobial production, an improvement instimulating the production of B cells, an improvement in stimulating theproduction of lymphocytes, an improvement in length of villi, and animprovement in expression of inflammatory cytokines; and/or wherein atleast one of the one or more bacteria and/or at least one of the one ormore fungi is desiccated.
 21. The composition of claim 20, wherein thefowl is a broiler.
 22. The composition of claim 20, wherein the one ormore bacteria and/or the one or more fungi have a MIC score of at leastabout 0.1.
 23. The composition of claim 20 wherein the purifiedmicrobial population comprises a bacterium with a 16S nucleic acidsequence sharing at least 97% identity with SEQ ID NO:386.
 24. Thecomposition of claim 20, wherein the purified microbial populationcomprises a bacterium with a 16S nucleic acid sequence sharing at least97% identity with SEQ ID NO:387.
 25. The composition of claim 23,wherein the purified microbial population further comprises one or morebacteria with a 16S nucleic acid sequence sharing at least 97% sequenceidentity with nucleic acid sequences selected from the group consistingof SEQ ID NO:13, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:369,SEQ ID NO:374, and SEQ ID NO:348.
 26. The composition of claim 25,wherein the purified microbial population comprises one or more bacteriawith a 16S nucleic acid sequence sharing at least 97% sequence identitywith SEQ ID NO:386 and SEQ ID NO:387.
 27. The composition of claim 26,wherein the purified microbial population comprises one or more bacteriawith a 16S nucleic acid sequence comprising SEQ ID NO:386 and SEQ IDNO:387.
 28. The composition of claim 25, wherein the purified microbialpopulation comprises one or more bacteria with a 16S nucleic acidsequence selected from the group consisting of SEQ ID NO:13, SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:369, SEQ ID NO:374, and SEQID NO:348.
 29. The composition of claim 23, wherein the purifiedmicrobial population further comprises one or more bacteria with a 16Snucleic acid sequence sharing at least 97% sequence identity withnucleic acid sequences selected from the group consisting of SEQ ID NO:1and SEQ ID NO:3.
 30. The composition of claim 29, wherein the purifiedmicrobial population comprises one or more bacteria with a 16S nucleicacid sequence sharing at least 97% sequence identity with SEQ ID NO:386and SEQ ID NO:387.
 31. The composition of claim 30, wherein the purifiedmicrobial population comprises one or more bacteria with a 16S nucleicacid sequence comprising SEQ ID NO:386 and SEQ ID NO:387.
 32. Thecomposition of claim 29, wherein the purified microbial populationcomprises one or more bacteria with a 16S nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:1 and SEQ ID NO:3.
 33. Thecomposition of claim 20, wherein the microbial composition is formulatedas an encapsulation, tablet, capsule, pill, feed additive, foodingredient, food preparation, food supplement, water additive,water-mixed additive, heat-stabilized additive, moisture-stabilizedadditive, pre-pelleted feed additive, pelleted feed additive,post-pelleting-applied feed additive, consumable solution, consumablespray additive, consumable solid, consumable gel, injection,suppository, drench, bolus, or combination thereof.
 34. The compositionof claim 20, wherein the improvement of the one or more desirable traitsis an improvement in antimicrobial production in the gastrointestinaltract.
 35. The composition of claim 20, wherein the microbialcomposition elicits a decrease in the variability of the number ofunique species in the gastrointestinal tract.
 36. The composition ofclaim 35, wherein the decrease in the variability of the number ofunique species is a reduction of the total number of unique species ofmicrobes to between about 50 and 400 species.
 37. The method of claim 5,wherein the purified microbial population further comprises one or morebacteria with a 16S nucleic acid sequence sharing at least 97% sequenceidentity with nucleic acid sequences selected from the group consistingof SEQ ID NO:13, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:369,SEQ ID NO:374, and SEQ ID NO:348.
 38. The method of claim 5, wherein thepurified microbial population further comprises one or more bacteriawith a 16S nucleic acid sequence sharing at least 97% sequence identitywith nucleic acid sequences selected from the group consisting of SEQ IDNO:1 and SEQ ID NO:3.
 39. The composition of claim 24, wherein thepurified microbial population further comprises one or more bacteriawith a 16S nucleic acid sequence sharing at least 97% sequence identitywith nucleic acid sequences selected from the group consisting of SEQ IDNO:13, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:31, SEQ ID NO:369, SEQ IDNO:374, and SEQ ID NO:348.
 40. The composition of claim 24, wherein thepurified microbial population further comprises one or more bacteriawith a 16S nucleic acid sequence sharing at least 97% sequence identitywith nucleic acid sequences selected from the group consisting of SEQ IDNO:1 and SEQ ID NO:3.